Alpha-amylase and alpha-amylase variants

ABSTRACT

The invention relates to a novel Termamyl-like alpha-amylase, and Termamyl-like alpha-amylases comprising mutations in two, three, four, five or six regions/positions. The variants have increased thermostability at acidic pH and/or at low Ca 2+  concentrations (relative to the parent). The invention also relates to a DNA construct comprising a DNA sequence encoding an alpha-amylase variant of the invention, a recombinant expression vector which carries a DNA construct of the invention, a cell which is transformed with a DNA construct of the invention, the use of an alpha-amylase variant of the invention for washing and/or dishwashing, textile desizing, starch liquefaction, a detergent additive comprising an alpha-amylase variant of the invention, a manual or automatic dishwashing detergent composition comprising an alpha-amylase variant of the invention, a method for generating a variant of a parent Termamyl-like alpha-amylase, which variant exhibits increased thermostability at acidic pH and/or at low Ca 2+  concentrations (relative to the parent).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 14/079,118 filed Nov. 13, 2013, pending, which is a divisional of U.S. application Ser. No. 13/556,623 filed Jul. 24, 2012, now abandoned, which is a division of U.S. application Ser. No. 12/493,860 filed Jun. 29, 2009 (now U.S. Pat. No. 8,263,368), which is a divisional of U.S. application Ser. No. 10/327,837 filed Dec. 23, 2002 (now U.S. Pat. No. 7,566,561), which is a divisional of U.S. application Ser. No. 09/545,586 filed on Apr. 7, 2000 (now U.S. Pat. No. 6,528,298), which is a divisional of U.S. application Ser. No. 09/290,734 filed on Apr. 13, 1999 (now U.S. Pat. No. 6,361,989), which is a continuation-in-part of U.S. application Ser. No. 09/170,670 filed on Oct. 13, 1998 (now U.S. Pat. No. 6,187,576), and claims priority under 35 U.S.C. 119 of Danish application nos. 1172/97 and PA 1999 00439 filed on Oct. 13, 1997 and Mar. 31, 1999, respectively, and U.S. provisional application No. 60/063,306 filed on Oct. 28, 1997, the contents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates a novel alpha-amylase within the family of Termamyl-like alpha-amylases suitable for detergents. The invention also relates to variants (mutants) of parent Termamyl-like alpha-amylases, notably variants exhibiting increased thermostability at acidic pH and/or at low Ca²⁺ concentrations (relative to the parent) which are advantageous with respect to applications of the variants in, industrial starch processing particularly (e.g., starch liquefaction or saccharification). Said alpha-amylase and alpha-amylase variants of the invention may advantageously also be used in detergents.

BACKGROUND OF THE INVENTION

Alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute a group of enzymes which catalyze hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides.

There is a very extensive body of patent and scientific literature relating to this industrially very important class of enzymes.

A number of alpha-amylase such as Termamyl-like alpha-amylases variants are known from, e.g., WO 90/11352, WO 95/10603, WO 95/26397, WO 96/23873 and WO 96/23874.

Among more recent disclosures relating to alpha-amylases, WO 96/23874 provides three-dimensional, X-ray crystal structural data for a Termamyl-like alpha-amylase which consists of the 300 N-terminal amino acid residues of the B. amyloliquefaciens alpha-amylase and amino acids 301-483 of the C-terminal end of the B. licheniformis alpha-amylase comprising the amino acid sequence (the latter being available commercially under the tradename Termamyl™), and which is thus closely related to the industrially important Bacillus alpha-amylases (which in the present context are embraced within the meaning of the term “Termamyl-like alpha-amylases”, and which include, inter alfa, the B. licheniformis, B. amyloliquefaciens and B. stearothermophilus alpha-amylases). WO 96/23874 further describes methodology for designing, on the basis of an analysis of the structure of a parent Termamyl-like alpha-amylase, variants of the parent Termamyl-like alpha-amylase which exhibit altered properties relative to the parent.

WO 95/35382 (Gist Brocades B.V.) concerns amylolytic enzymes derived from B. licheniformis with improved properties allowing reduction of the Ca²⁺ concentration under application without a loss of performance of the enzyme. The amylolytic enzyme comprises one or more amino acid changes at positions selected from the group of 104, 128, 187, 188 of the B. licheniformis alpha-amylase sequence.

WO 96/23873 (Novo Nordisk) discloses Termamyl-like alpha-amylase variants which have increased thermostability obtained by pairwise deletion in the region R181*, G182*, T183* and G184* of the sequence shown in SEQ ID NO: 1 herein.

WO 97/00324 (KAO) discloses a gene encoding an alkaline liquefying alpha-amylase derived from Bacillus sp. strain KSM-AP1378 with the deposited no. FERM BP-3048 suitable for detergents.

BRIEF DISCLOSURE OF THE INVENTION

The present invention relates to a novel alpha-amylase and to novel alpha-amylolytic variants (mutants) of a Termamyl-like alpha-amylase, in particular variants exhibiting increased thermostability (relative to the parent) which are advantageous in connection with the industrial processing of starch (starch liquefaction, saccharification and the like). The novel alpha-amylase is suitable for laundry washing and dishwash as is has a high activity under wash conditions at alkaline pHs in the range 9-11.

The inventors have surprisingly found out that in case of combining two, three, four, five or six mutations (will be described below), the thermostability of Termamyl-like alpha-amylases is increased at acidic pH and/or at low Ca²⁺ concentration in comparison to single mutations, such as the mutation disclosed in WO 96/23873 (Novo Nordisk), i.e., pairwise deletion in the region R181*, G182*, T183* and G184* of the sequence shown in SEQ ID NO: 1 herein.

The invention further relates to DNA constructs encoding variants of the invention, to composition comprising variants of the invention, to methods for preparing variants of the invention, and to the use of variants and compositions of the invention, alone or in combination with other alpha-amylolytic enzymes, in various industrial processes, e.g., starch liquefaction.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1, 1A and B is an alignment of the amino acid sequences of nine parent Termamyl-like alpha-amylases. The numbers on the Extreme left designate the respective amino acid sequences as follows:

1: SEQ ID NO: 2,

2: Bacillus sp. strain KSM-AP1378 disclosed in WO 97/00324

3: SEQ ID NO: 1, 4: SEQ ID NO: 5, 5: SEQ ID NO: 4, 6: SEQ ID NO: 3.

7: Partial alpha-amylase sequence

8: SEQ ID NO: 24 9: SEQ ID NO: 26.

FIG. 2 shows the pH Profile of the AA560 alpha-amylase compared to the SP722 and SP690 alpha-amylases. The pH profile was measured at 37° C. The activity is shown in absolute values as Abs650/mg.

FIG. 3 shows the Temperature Profile of the AA560 alpha-amylase compared to the SP722 and SP690 alpha-amylases. The temperature profile shown as Abs650/mg.

FIG. 4 shows the wash performance of AA560 in the AP Model Detergent 97 in comparison to SP722, SP690 and Termamyl®.

FIG. 5 shows the wash performance of AA560 in the Omo Multi Acao in comparison to SP722, SP690 and Termamyl®.

FIG. 6 shows the wash performance of AA560 in the Omo Concentrated in comparison to SP722, SP690 and Termamyl®.

FIG. 7 shows the wash performance of AA560 in the Ariel Futur liquid in comparison to SP722, SP690 and Termamyl®.

DETAILED DISCLOSURE OF THE INVENTION Alpha-Amylase Activity Determination

Alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute a group of enzymes which catalyze hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides. For purposes of the present invention, alpha-amylase activity may be determined using the Phadebas assay, the pNPG7 assay and the BS-alpha-amylase activity assay described below in the “Materials and Methods” section.

The Novel Alpha-Amylase Microbial Source

The novel alkaline alpha-amylase of the invention may be derived from a strain of Bacillus. Preferred strains are of Bacillus sp. DSM 12649 (the AA560 alpha-amylase) or Bacillus sp. DSM 12648 (the AA349 alpha-amylase). These strains were deposited on 25 Jan. 1999 by the inventors under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at Deutshe Sammmlung von Microorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig DE.

Escherichia coli strains termed NN049467 and NN049470 containing the alpha-amylase genes in plasmids pLiH1274 (AA349) and plasmid pTVB299 (AA560) have also been deposited on 7 Apr. 1999 under the terms of the Budapest Treaty with the Deutshe Sammmlung von Microorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg 1 b, D-38124 Braunschweig DE, and given the accession numbers DSM12761 and DSM12764, respectively.

Homology of Enzyme

In a first embodiment, the present invention relates to isolated polypeptides having an amino acid sequence which has a degree of identity to amino acids 1 to 485 of SEQ ID NO: 24 or SEQ ID NO: 26 (i.e., the mature polypeptide) of at least about 96%, preferably at least about 97%, more preferably at least about 98%, even more preferably at least about 99%, which have alpha-amylase activity (hereinafter “homologous polypeptides”). In a preferred embodiment, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from amino acids 1 to 485 of SEQ ID NO: 24 or SEQ ID NO: 26. It is to be noted that SEQ ID NO: 24 and SEQ ID NO: 26 are identical. However, the DNA sequences, i.e., SEQ ID NO: 23 and SEQ ID NO: 25, respectively, encoding the alpha-amylase of the invention shown in SEQ ID NO: 24 and SEQ ID NO: 26 are not identical.

The amino acid sequence homology may be determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art. Thus, GAP provided in GCG version 8 (Needleman and Wunsch, 1970, Journal of Molecular Biology 48, 443-453) may be used for a pairwise alignment of the sequences and calculation of the degree of identity or degree of homology using the default settings. Alternatively, Gap from GCG version 9 may be used with a translated version 8 peptide scoring matrix, a gap creation penalty of 30, a gap extension penalty of 1 using ntol's matrix (http://plasmid/˜bioweb/matrix/) without end gap penalty.

Homology (Identity) of the Novel Alpha-Amylase to Known Bacillus sp. Alpha-Amylases

A homology search of known sequences showed homologies for the sequences of the invention with a number of Bacillus amylases in the range 65-95% on amino acid basis determined as described above.

Specifically, the most homologous alpha-amylases found are SP690 (SEQ ID NO: 1 of U.S. Pat. No. 5,856,164 which is about 87% homologous), SP722 (SEQ ID NO: 2 of U.S. Pat. No. 5,856,164 which is about 87% homologous), the mature part (i.e., amino acids no. 31-516) of the alpha-amylase obtained from Bacillus sp. KSM-AP1378 disclosed as SEQ ID NO: 2 of WO 97/00324 which is about 86% homologous, and the alpha-amylase disclosed in Tsukamoto et al., 1988, Biochem. Biophys. Res. Commun. 151: 25-33) which is about 95% homologous to SEQ ID NO: 24 and SEQ ID NO: 26 determined as describe above.

Preferably, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26 or allelic variants thereof; or fragments thereof that have alpha-amylase activity. SEQ ID NO: 24 and SEQ ID NO: 26 show the mature part of the alkaline alpha-amylase of the invention.

A fragment of SEQ ID NO: 24 or SEQ ID NO: 26 are polypeptides having one or more amino acids deleted from the amino and/or carboxyl terminus of this amino acid sequence.

An allelic variant denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

The amino acid sequences of the homologous polypeptides may differ from the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26 by an insertion or deletion of one or more amino acid residues and/or the substitution of one or more amino acid residues by different amino acid residues. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions which do not generally alter the specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as these in reverse.

In a second embodiment, the present invention relates to isolated polypeptides having alpha-amylase activity which are encoded by nucleic acid sequences which hybridize under medium stringency conditions, preferably medium-high stringency conditions, more preferably high stringency conditions, and most preferably very high stringency conditions with a nucleic acid probe which hybridizes under the same conditions with (i) the nucleic acid sequence of SEQ ID NO: 23 or SEQ ID NO: 25, (ii) the cDNA sequence of SEQ ID NO: 23 or SEQ ID NO: 25, (iii) a subsequence of (i) or (ii), or (iv) a complementary strand of (i), (ii), or (iii) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). The subsequence of SEQ ID NO: 23 or SEQ ID NO: 25 may be at least 100 nucleotides or preferably at least 200 nucleotides. Moreover, the subsequence may encode a polypeptide fragment which has alpha-amylase activity. The polypeptides may also be allelic variants or fragments of the polypeptides that have alpha-amylase activity.

The nucleic acid sequence of SEQ ID NO: 23 or SEQ ID NO: 25 or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26 or a fragment thereof, may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having alpha-amylase activity from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, preferably at least 25, and more preferably at least 35 nucleotides in length. Longer probes can also be used. Both DNA and RNA probes can be used. The probes are typically labelled for detecting the corresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed by the present invention.

Thus, a genomic DNA or cDNA library prepared from such other organisms may be screened for DNA which hybridizes with the probes described above and which encodes a polypeptide having alpha-amylase activity. Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA which is homologous with SEQ ID NO: 23 or SEQ ID NO: 25 or subsequences thereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that the nucleic acid sequence hybridizes to a nucleic acid probe corresponding to the nucleic acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 25, its complementary strand, or subsequences thereof, under medium to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions are detected using X-ray film.

In another preferred embodiment, the nucleic acid probe is the nucleic acid sequence contained in plasmids pLiH1274 (AA349) or pTVB299 (AA560) which are contained in Escherichia coli DSM12761 or Escherichia coli DSM12764, respectively, or, wherein the nucleic acid sequence encodes a polypeptide having acid alpha-amylase activity of the invention and shown in SEQ ID NO: 24 and SEQ ID NO: 26, respectively.

For long probes of at least 100 nucleotides in length, medium to very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 ig/ml sheared and denatured salmon sperm DNA, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures.

For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS preferably at least at 55° C. (medium stringency), preferably at least at 60° C. (medium-high stringency), more preferably at least at 65° C. (high stringency), and most preferably at least at 70° C. (very high stringency).

For short probes which are about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization, hybridization, and washing post-hybridization at 5° C. to 10° C. below the calculated T_(m) using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, lx Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed once in 6×SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C. below the calculated T_(m).

In a third embodiment, the present invention relates to isolated polypeptides, i.e., the polypeptides shown in SEQ ID NO: 24 or SEQ ID NO: 26, having the following physicochemical properties:

A pH optimum (see FIG. 2) determined using the Phadebas method (37° C.) was found to be in the range between pH 8 and 9, more precisely at about 8.5.

A temperature optimum (See FIG. 3) determined using the Phasebas method (pH 9.0) was found to be in the range between 55 and 65° C., more precisely about 60° C.

A pI between 7-8 (See Table 1 in Example 11) was determined by isoelectric focusing (Pharmacia, Ampholine, pH 3.5-9.3).

A specific activity (see Table 1 of Example 11) of 35,000 NU/ml was determined using the Phadebas method and 6,000 NU/ml using the pNPG7 method.

The alpha-amylase of the present invention have at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 100% of the alpha-amylase activity of the mature alpha-amylase shown in SEQ ID NO: 24 and SEQ ID NO: 26.

An alpha-amylase of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the alpha-amylase encoded by the nucleic acid sequence is produced by the source or by a cell in which the nucleic acid sequence from the source has been inserted.

An alpha-amylase of the present invention is a bacterial polypeptide. For example, the polypeptide may be a gram positive bacterial polypeptide such as a Bacillus polypeptide, e.g., a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide; or a Streptomyces polypeptide, e.g., a Streptomyces lividans or Streptomyces murinus polypeptide; or a gram negative bacterial polypeptide, e.g., an E. coli or a Pseudomonas sp. polypeptide.

In another preferred embodiment, the polypeptide is a Bacillus sp. polypeptide, more preferred embodiment, the polypeptide is a Bacillus sp. DSM 12648 or Bacillus sp. DSM 12649 polypeptide, e.g., the polypeptides with the amino acid sequence of SEQ ID NO: 24 and SEQ ID NO: 26, respectively.

It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art. The nucleic acid sequence may then be derived by similarly screening a genomic or cDNA library of another microorganism. Once a nucleic acid sequence encoding a polypeptide has been detected with the probe(s), the sequence may be isolated or cloned by utilizing techniques which are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

As defined herein, an “isolated” polypeptide is a polypeptide which is essentially free of other non-alpha-amylase polypeptides, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by SDS-PAGE.

Polypeptides encoded by nucleic acid sequences of the present invention also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding another polypeptide to a nucleic acid sequence (or a portion thereof) of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator.

Mutants of the Novel Alpha-Amylase

Specifically contemplated mutants of the novel alpha-amylase shown in SEQ ID NO: 24 (or SEQ ID NO: 26) are described in the following. A mutant alpha-amylase of the invention is characterized by the fact that one or more of the methionine amino acid residues is exhanged with any amino acid residue except for Cys and Met. Thus, according to the invention the amino acid residues to replace the methionine amino acid residue are the following: Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

A preferred embodiment of the mutant alpha-amylase of the invention is characterized by the fact that one or more of the methionine amino acid residues is (are) exchanged with a Leu, Thr, Ala, Gly, Ser, Ile, or Val amino acid residue, preferably a Leu, Thr, Ala, or Gly amino acid residue. In this embodiment a very satisfactory activity level and stability in the presence of oxidizing agents is obtained. Specifically this means that one or more of the methiones in the following position may be replaced or deleted using any suitable technique known in the art, including especially site directed mutagenesis and gene shuffling. Contemplated position, using the SEQ ID NO: 24 numbering, are: 9, 10, 105, 116, 202, 208, 261, 309, 323, 382, 430, 440.

In a preferred embodiment of the mutant alpha-amylase of the invention is characterized by the fact that the methionine amino acid residue at position 202 is exchanged with any of amino acid residue expect for Cys and Met, preferably with a Leu, Thr, Ala, Gly, Ser, Ile, or Asp.

Other contemplated preferred mutations include deletion of one, two or more residues of amino acids R181, G182, D183 or G184, K185, G186 or substitution of one or more of these residues. A preferred mutation is the deletion of D183-G184. Particularly relevant mutations are substitutions of G186 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. A particularly preferred substitution is G186R.

Also contemplated is substitution of N195 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. A particularly interesting substitution is N195F.

The following combinations of the above metioned mutations include: deletion of D183-G184+N195F, deletion of D183-G184+G186R, deletion of D183-G184+G186R+N195F and G186R+N195F.

Nucleic Acid Sequences

The present invention also relates to isolated nucleic acid sequences which encode a polypeptide of the present invention. In a preferred embodiment, the nucleic acid sequence is set forth in SEQ ID NO: 23 or SEQ ID NO: 25. In another more preferred embodiment, the nucleic acid sequence is the sequence contained in plasmid pLiH1274 (AA349) or plasmid pTVB299 (AA560) that is contained in Escherichia coli DSM12761 and Escherichia coli DSM12764, respectively. In another preferred embodiment, the nucleic acid sequence is the mature polypeptide coding region of SEQ ID NO: 23 or SEQ ID NO: 25. The present invention also encompasses nucleic acid sequences which encode a polypeptide having the amino acid sequence of SEQ ID NO: 24 which differ from SEQ ID NO: 23 or SEQ ID NO: 25 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 23 or SEQ ID NO: 25 which encode fragments of SEQ ID NO: 24 or SEQ ID NO: 26, respectively, that have alpha-amylase activity.

Subsequences of SEQ ID NO: 23 or SEQ ID NO: 25 are nucleic acid sequences encompassed by SEQ ID NO: 23 or SEQ ID NO: 25 except that one or more nucleotides from the 5′ and/or 3′ end have been deleted.

The present invention also relates to mutant nucleic acid sequences comprising at least one mutation in the mature polypeptide coding sequence of SEQ ID NO:1 or SEQ ID NO: 3, in which the mutant nucleic acid sequence encodes a polypeptide which consists of amino acids 1 to 485 of SEQ ID NO: 24 or SEQ ID NO: 26.

The techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the nucleic acid sequences of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used. The nucleic acid sequence may be cloned from a strain of Bacillus, or another or related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleic acid sequence.

The term “isolated nucleic acid sequence” as used herein refers to a nucleic acid sequence which is essentially free of other nucleic acid sequences, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably at least about 60% pure, even more preferably at least about 80% pure, and most preferably at least about 90% pure as determined by agarose electrophoresis. For example, an isolated nucleic acid sequence can be obtained by standard cloning procedures used in genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated. The nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

Homology of DNA Sequence Encoding the Enzyme

The present invention also relates to nucleic acid sequences which have a degree of homology to the mature polypeptide coding sequence of SEQ ID NO: 23 (i.e., nucleotides 1 to 1458) or SEQ ID NO: 25 (i.e., nucleotide 1 to 1458) of at least about 96% homology on DNA level, preferably about 97%, preferably about 98%, more preferably about 99% homology, which encode an active polypeptide.

The DNA sequence homology may be determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (described above). Thus, Gap GCGv8 may be used with the following default parameters: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, default scoring matrix. GAP uses the method of Needleman/Wunsch/Sellers to make alignments.

Modification of a nucleic acid sequence encoding a polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term “substantially similar” to the polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., variants that differ in specific activity, thermostability, pH optimum, or the like. The variant sequence may be constructed on the basis of the nucleic acid sequence presented as the polypeptide encoding part of SEQ ID NO: 23 or SEQ ID NO: 25, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide encoded by the nucleic acid sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active polypeptide. Amino acid residues essential to the activity of the polypeptide encoded by the isolated nucleic acid sequence of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for [enzyme] activity to identify amino acid residues that are critical to the activity of the molecule. Sites of substrate-enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labelling (see, e.g., de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The present invention also relates to isolated nucleic acid sequences encoding a polypeptide of the present invention, which hybridize under medium stringency conditions, preferably medium-high stringency conditions, more preferably high stringency conditions, and most preferably very high stringency conditions with a nucleic acid probe which hybridizes under the same conditions with the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO: 3 or its complementary strand; or allelic variants and subsequences thereof (Sambrook et al., 1989, supra), as defined herein.

The present invention also relates to isolated nucleic acid sequences produced by (a) hybridizing a DNA under medium, medium-high, high, or very high stringency conditions with the sequence of SEQ ID NO: 23 or SEQ ID NO: 25, or their complementary strands, or a subsequence thereof; and (b) isolating the nucleic acid sequence. The subsequence is preferably a sequence of at least 100 nucleotides such as a sequence which encodes a polypeptide fragment which has alpha-amylase activity.

Methods for Producing Mutant Nucleic Acid Sequences

The present invention further relates to methods for producing a mutant nucleic acid sequence, comprising introducing at least one mutation into the mature polypeptide coding sequence of SEQ ID NO: 23 or SEQ ID NO: 25 or a subsequence thereof, wherein the mutant nucleic acid sequence encodes a polypeptide which consists of 1 to 485 of SEQ ID NO: 24 or SEQ ID NO: 26 or a fragment thereof which has alpha-amylase activity.

The introduction of a mutation into the nucleic acid sequence to exchange one nucleotide for another nucleotide may be accomplished by site-directed mutagenesis using any of the methods known in the art. Particularly useful is the procedure which utilizes a supercoiled, double stranded DNA vector with an insert of interest and two synthetic primers containing the desired mutation. The oligonucleotide primers, each complementary to opposite strands of the vector, extend during temperature cycling by means of Pfu DNA polymerase. On incorporation of the primers, a mutated plasmid containing staggered nicks is generated. Following temperature cycling, the product is treated with DpnI which is specific for methylated and hemimethylated DNA to digest the parental DNA template and to select for mutation-containing synthesized DNA. Other procedures known in the art may also be used. These other procedures include gene shuffling, e.g., as described in WO 95/22625 (from Affymax Technologies N.V.) and WO 96/00343 (from Novo Nordisk A/S).

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising a nucleic acid sequence of the present invention operably linked to one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

“Nucleic acid construct” is defined herein as a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence of the present invention. The term “coding sequence” is defined herein as a portion of a nucleic acid sequence which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by a ribosome binding site (prokaryotes) or by the ATG start codon (eukaryotes) located just upstream of the open reading frame at the 5′ end of the mRNA and a transcription terminator sequence located just downstream of the open reading frame at the 3′ end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

An isolated nucleic acid sequence encoding a polypeptide of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the nucleic acid sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleic acid sequences utilizing recombinant DNA methods are well known in the art.

The term “control sequences” is defined herein to include all components which are necessary or advantageous for the expression of a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide. The term “operably linked” is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the expression of a polypeptide.

Promoter Sequence

The control sequence may be an appropriate promoter sequence, a nucleic acid sequence which is recognized by a host cell for expression of the nucleic acid sequence. The promoter sequence contains transcriptional control sequences which mediate the expression of the polypeptide. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell, are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80: 21-25). Further promoters are described in “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Terminator Sequence

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′ terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

Signal Peptide

The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5′ end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide. Alternatively, the 5′ end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide. However, any signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention.

Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

Regulatory System

It may also be desirable to add regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those which allow for gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene which is amplified in the presence of methotrexate, and the metallothionein genes which are amplified with heavy metals. In these cases, the nucleic acid sequence encoding the polypeptide would be operably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectors comprising a nucleic acid sequence of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites. Alternatively, the nucleic acid sequence of the present invention may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.

The vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. A selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s) that permits stable integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome of the cell.

For integration into the host cell genome, the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus. Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75: 1433).

More than one copy of a nucleic acid sequence of the present invention may be inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

Host Cells

The present invention also relates to recombinant host cells, comprising a nucleic acid sequence of the invention, which are advantageously used in the recombinant production of the polypeptides. A vector comprising a nucleic acid sequence of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus, or gram negative bacteria such as E. coli and Pseudomonas sp. In a preferred embodiment, the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. In another preferred embodiment, the Bacillus cell is an alkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), using competent cells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771-5278).

Methods of Production

The present invention also relates to methods for producing an alpha-amylase of the present invention comprising (a) cultivating a strain, which in its wild-type form is capable of producing the polypeptide, to produce a supernatant comprising the polypeptide; and (b) recovering the polypeptide. Preferably, the strain is of the genus Bacillus sp.

The present invention also relates to methods for producing an alpha-amylase of the present invention comprising (a) cultivating a host cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

The present invention also relates to methods for producing an alpha-amylase of the present invention comprising (a) cultivating a host cell under conditions conducive for production of the polypeptide, wherein the host cell comprises a mutant nucleic acid sequence having at least one mutation in the mature polypeptide coding region of SEQ ID NO: 23 or SEQ ID NO: 25, wherein the mutant nucleic acid sequence encodes a polypeptide which consists of amino acids 1 to 485 of SEQ ID NO: 24 or SEQ ID NO: 26, and (b) recovering the polypeptide.

Mutant Alpha-Amylase

The present invention also relates to alpha-amylase mutants.

The Termamyl-Like Alpha-Amylase

It is well known that a number of alpha-amylases produced by Bacillus spp. are highly homologous on the amino acid level. For instance, the B. licheniformis alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 4 (commercially available as Termamyl™) has been found to be about 89% homologous with the B. amyloliquefaciens alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 5 and about 79% homologous with the B. stearothermophilus alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 3. Further homologous alpha-amylases include an alpha-amylase derived from a strain of the Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in detail in WO 95/26397, and the alpha-amylase described by Tsukamoto et al., Biochemical and Biophysical Research Communications, 151 (1988), pp. 25-31. Also the novel alpha-amylase of the invention, of which a specific embodiment is shown in SEQ ID NO: 24 (and SEQ ID NO: 26), is contemplated as the parent alpha-amylase to be mutated according to the invention.

Still further homologous alpha-amylases include the alpha-amylase produced by the B. licheniformis strain described in EP 0252666 (ATCC 27811), and the alpha-amylases identified in WO 91/00353 and WO 94/18314. Other commercial Termamyl-like B. licheniformis alpha-amylases are Duramyl™ from Novo Nordisk, Optitherm™ and Takatherm™ (available from Solvay), Maxamyl™ (available from Gist-brocades/Genencor), Spezym AA™ and Spezyme Delta AA™ (available from Genencor), and Keistase™ (available from Daiwa).

Because of the substantial homology found between these alpha-amylases, they are considered to belong to the same class of alpha-amylases, namely the class of “Termamyl-like alpha-amylases”.

Accordingly, in the present context, the term “Termamyl-like alpha-amylase” is intended to indicate an alpha-amylase which, at the amino acid level, exhibits a substantial homology to Termamyl™, i.e., the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 4 herein. In other words, a Termamyl-like alpha-amylase is an alpha-amylase which has the amino acid sequence shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 or 8 herein, and the amino acid sequence shown in SEQ ID NO: 1 of WO 95/26397 (the same as the amino acid sequence shown as SEQ ID NO: 7 herein) or in SEQ ID NO: 2 of WO 95/26397 (the same as the amino acid sequence shown as SEQ ID NO: 8 herein) or in Tsukamoto et al., 1988, (which amino acid sequence is shown in SEQ ID NO: 6 herein) or i) which displays at least 60%, preferred at least 70%, more preferred at least 75%, even more preferred at least 80%, especially at least 85%, especially preferred at least 90%, even especially more preferred at least 95% homology with at least one of said amino acid sequences shown in SEQ ID NOS: 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 and/or ii) displays immunological cross-reactivity with an antibody raised against at least one of said alpha-amylases, and/or iii) is encoded by a DNA sequence which hybridizes to the DNA sequences encoding the above-specified alpha-amylases which are apparent from SEQ ID NOS: 9, 10, 11, 12 or 13 of the present application (which encoding sequences encode the amino acid sequences shown in SEQ ID NOS: 1, 2, 3, 4 and 5 herein, respectively), from SEQ ID NO: 4 of WO 95/26397 (which DNA sequence, together with the stop codon TAA, is shown in SEQ ID NO: 14 herein and encodes the amino acid sequence shown in SEQ ID NO: 8 herein) and from SEQ ID NO: 5 of WO 95/26397 (shown in SEQ ID NO: 15 herein), respectively.

In connection with property i), the “homology” may be determined by use of any conventional algorithm, preferably by use of the GAP progamme from the GCG package version 7.3 (June 1993) using default values for GAP penalties, which is a GAP creation penalty of 3.0 and GAP extension penalty of 0.1, (Genetic Computer Group (1991) Programme Manual for the GCG Package, version 7, 575 Science Drive, Madison, Wis., USA 53711).

A structural alignment between Termamyl and a Termamyl-like alpha-amylase may be used to identify equivalent/corresponding positions in other Termamyl-like alpha-amylases. One method of obtaining said structural alignment is to use the Pile Up programme from the GCG package using default values of gap penalties, i.e., a gap creation penalty of 3.0 and gap extension penalty of 0.1. Other structural alignment methods include the hydrophobic cluster analysis (Gaboriaud et al., 1987, FEBS Letters 224:149-155) and reverse threading (Huber and Torda, 1998, Protein Science 7(1): 142-149.

Property ii) of the alpha-amylase, i.e., the immunological cross reactivity, may be assayed using an antibody raised against, or reactive with, at least one epitope of the relevant Termamyl-like alpha-amylase. The antibody, which may either be monoclonal or polyclonal, may be produced by methods known in the art, e.g., as described by Hudson et al., Practical Immunology, Third edition (1989), Blackwell Scientific Publications. The immunological cross-reactivity may be determined using assays known in the art, examples of which are Western Blotting or radial immunodiffusion assay, e.g., as described by Hudson et al., 1989. In this respect, immunological cross-reactivity between the alpha-amylases having the amino acid sequences SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8 respectively, have been found.

The oligonucleotide probe used in the characterization of the Termamyl-like alpha-amylase in accordance with property iii) above may suitably be prepared on the basis of the full or partial nucleotide or amino acid sequence of the alpha-amylase in question.

Suitable conditions for testing hybridization involve presoaking in 5×SSC and prehybridizing for 1 hour at ˜40° C. in a solution of 20% formamide, 5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 mg of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 100 mM ATP for 18 hours at ˜40° C., followed by three times washing of the filter in 2×SSC, 0.2% SDS at 40° C. for 30 minutes (low stringency), preferred at 50° C. (medium stringency), more preferably at 65° C. (high stringency), even more preferably at ˜75° C. (very high stringency). More details about the hybridization method can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.

In the present context, “derived from” is intended not only to indicate an alpha-amylase produced or producible by a strain of the organism in question, but also an alpha-amylase encoded by a DNA sequence isolated from such strain and produced in a host organism transformed with said DNA sequence. Finally, the term is intended to indicate an alpha-amylase which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the alpha-amylase in question. The term is also intended to indicate that the parent alpha-amylase may be a variant of a naturally occurring alpha-amylase, i.e., a variant which is the result of a modification (insertion, substitution, deletion) of one or more amino acid residues of the naturally occurring alpha-amylase.

Parent Hybrid Alpha-Amylases

The parent alpha-amylase may be a hybrid alpha-amylase, i.e., an alpha-amylase which comprises a combination of partial amino acid sequences derived from at least two alpha-amylases.

The parent hybrid alpha-amylase may be one which on the basis of amino acid homology and/or immunological cross-reactivity and/or DNA hybridization (as defined above) can be determined to belong to the Termamyl-like alpha-amylase family. In this case, the hybrid alpha-amylase is typically composed of at least one part of a Termamyl-like alpha-amylase and part(s) of one or more other alpha-amylases selected from Termamyl-like alpha-amylases or non-Termamyl-like alpha-amylases of microbial (bacterial or fungal) and/or mammalian origin.

Thus, the parent hybrid alpha-amylase may comprise a combination of partial amino acid sequences deriving from at least two Termamyl-like alpha-amylases, or from at least one Termamyl-like and at least one non-Termamyl-like bacterial alpha-amylase, or from at least one Termamyl-like and at least one fungal alpha-amylase. The Termamyl-like alpha-amylase from which a partial amino acid sequence derives may, e.g., be any of those specific Termamyl-like alpha-amylases referred to herein.

For instance, the parent alpha-amylase may comprise a C-terminal part of an alpha-amylase derived from a strain of B. licheniformis, and a N-terminal part of an alpha-amylase derived from a strain of B. amyloliquefaciens or from a strain of B. stearothermophilus. For instance, the parent alpha-amylase may comprise at least 430 amino acid residues of the C-terminal part of the B. licheniformis alpha-amylase, and may, e.g., comprise a) an amino acid segment corresponding to the 37 N-terminal amino acid residues of the B. amyloliquefaciens alpha-amylase having the amino acid sequence shown in SEQ ID NO: 5 and an amino acid segment corresponding to the 445 C-terminal amino acid residues of the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 4, or b) an amino acid segment corresponding to the 68 N-terminal amino acid residues of the B. stearothermophilus alpha-amylase having the amino acid sequence shown in SEQ ID NO: 3 and an amino acid segment corresponding to the 415 C-terminal amino acid residues of the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 4.

The non-Termamyl-like alpha-amylase may, e.g., be a fungal alpha-amylase, a mammalian or a plant alpha-amylase or a bacterial alpha-amylase (different from a Termamyl-like alpha-amylase). Specific examples of such alpha-amylases include the Aspergillus oryzae TAKA alpha-amylase, the A. niger acid alpha-amylase, the Bacillus subtilis alpha-amylase, the porcine pancreatic alpha-amylase and a barley alpha-amylase. All of these alpha-amylases have elucidated structures which are markedly different from the structure of a typical Termamyl-like alpha-amylase as referred to herein.

The fungal alpha-amylases mentioned above, i.e., derived from A. niger and A. oryzae, are highly homologous on the amino acid level and generally considered to belong to the same family of alpha-amylases. The fungal alpha-amylase derived from Aspergillus oryzae is commercially available under the tradename Fungamyl™

Furthermore, when a particular variant of a Termamyl-like alpha-amylase (variant of the invention) is referred to—in a conventional manner—by reference to modification (e.g., deletion or substitution) of specific amino acid residues in the amino acid sequence of a specific Termamyl-like alpha-amylase, it is to be understood that variants of another Termamyl-like alpha-amylase modified in the equivalent position(s) (as determined from the best possible amino acid sequence alignment between the respective amino acid sequences) are encompassed thereby.

A preferred embodiment of a variant of the invention is one derived from a B. licheniformis alpha-amylase (as parent Termamyl-like alpha-amylase), e.g., one of those referred to above, such as the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 4.

Construction of Variants of the Invention

The construction of the variant of interest may be accomplished by cultivating a microorganism comprising a DNA sequence encoding the variant under conditions which are conducive for producing the variant. The variant may then subsequently be recovered from the resulting culture broth. This is described in detail further below.

Altered Properties of Variants of the Invention

The following discusses the relationship between mutations which may be present in variants of the invention, and desirable alterations in properties (relative to those a parent, Termamyl-like alpha-amylase) which may result therefrom.

Increased Thermostability at Acidic pH and/or at Low Ca²⁺ Concentration

Mutations of particular relevance in relation to obtaining variants according to the invention having increased thermostability at acidic pH and/or at low Ca²⁺ concentration include mutations at the following positions (relative to B. licheniformis alpha-amylase, SEQ ID NO: 4): H156, N172, A181, N188, N190, H205, D207, A209, A210, E211, Q264, N265.

In the context of the invention the term “acidic pH” means a pH below 7.0, especially below the pH range, in which industrial starch liquefaction processes are normally performed, which is between pH 5.5 and 6.2.

In the context of the present invention the term “low Calcium concentration” means concentrations below the normal level used in industrial starch liquefaction. Normal concentrations vary depending of the concentration of free Ca²⁺ in the corn. Normally a dosage corresponding to 1 mM (40 ppm) is added which together with the level in corn gives between 40 and 60 ppm free Ca²⁺.

In the context of the invention the term “high tempertatures” means temperatures between 95° C. and 160° C., especially the temperature range in which industrial starch liquefaction processes are normally performed, which is between 95° C. and 105° C.

The inventors have now found that the thermostability at acidic pH and/or at low Ca²⁺ concentration may be increased even more by combining certain mutations including the above mentioned mutations and/or I201 with each other.

Said “certain” mutations are the following (relative to B. licheniformis alpha-amylase, SEQ ID NO: 4):

N190, D207, E211, Q264 and I201.

Said mutation may further be combined with deletions in one, preferably two or even three positions as described in WO 96/23873 (i.e., in positions R181, G182, T183, G184 in SEQ ID NO: 1 herein). According to the invention variants of a parent Termamyl-like alpha-amylase with alpha-amylase activity comprising mutations in two, three, four, five or six of the above positions are contemplated.

It should be emphasized that not only the Termamyl-like alpha-amylases mentioned specifically below are contemplated. Also other commercial Termamyl-like alpha-amylases are contemplated. An unexhaustive list of such alpha-amylases is the following: alpha-amylases produced by the B. licheniformis strain described in EP 0252666 (ATCC 27811), and the alpha-amylases identified in WO 91/00353 and WO 94/18314. Other commercial Termamyl-like B. licheniformis alpha-amylases are Optitherm™ and Takatherm™ (available from Solvay), Maxamyl™ (available from Gist-brocades/Genencor), Spezym AA™ Spezyme Delta AA™ (available from Genencor), and Keistase™ (available from Daiwa).

It may be mentioned here that amino acid residues, respectively, at positions corresponding to N190, I201, D207 and E211, respectively, in SEQ ID NO: 4 constitute amino acid residues which are conserved in numerous Termamyl-like alpha-amylases. Thus, for example, the corresponding positions of these residues in the amino acid sequences of a number of Termamyl-like alpha-amylases which have already been mentioned (vide supra) are as follows:

TABLE 1 Termamyl-like alpha-amylase N I D E Q B. licheniformis N190 I201 D207 E211 Q264 (SEQ ID NO: 4) B. amyloliquefaciens N190 V201 D207 E211 Q264 (SEQ ID NO: 5) B. stearothermophilus N193 L204 E210 E214 — (SEQ ID NO: 3) Bacillus WO 95/26397 N195 V206 E212 E216 — (SEQ ID NO: 2) Bacillus WO 95/26397 N195 V206 E212 E216 — (SEQ ID NO: 1) “Bacillus sp. #707” N195 I206 E212 E216 — (SEQ ID NO: 6) Bacillus sp. AA560 N195 I206 E212 E216 — (SEQ ID NO: 24)

Mutations of these conserved amino acid residues are very important in relation to improving thermostability at acidic pH and/or at low calcium concentration, and the following mutations are of particular interest in this connection (with reference to the numbering of the B. licheniformis amino acid sequence shown in SEQ ID NO: 4).

Pair-wise amino acid deletions at positions corresponding to R179-G182 in SEQ ID NO: 5 corresponding to a gap in SEQ ID NO: 4. when aligned with a numerous Termamyl-like alpha-amylases. Thus, for example, the corresponding positions of these residues in the amino acid sequences of a number of Termamyl-like alpha-amylases which have already been mentioned (vide supra) are as follows:

TABLE 2 Termamyl-like alpha-amylase Pair wise amino acid deletions among B. amyloliquefaciens R176, G177, E178, G179 (SEQ ID NO: 5) B. stearothermophilus R179, G180, I181, G182 (SEQ ID NO: 3) Bacillus WO 95/26397 R181, G182, T183, G184 (SEQ ID NO: 2) Bacillus WO 95/26397 R181, G182, D183, G184 (SEQ ID NO: 1) Bacillus sp. #707” R181, G182, H183, G184 (SEQ ID NO: 6) Bacillus sp. (AA560) R181, G182, H183, G184 (SEQ ID NO: 24)

When using SEQ ID NO: 1-6 or SEQ ID NO: 24 (or SEQ ID NO: 26) as the backbone (i.e., as the parent Termamyl-like alpha-amylase) two, three, four, five or six mutations may according to the invention be made in the following regions/positions to increase the thermostability at acidic pH and/or at low Ca²⁺ concentrations (relative to the parent):

(relative to SEQ ID NO: 1 herein):

1: R181*,G182*,T183*,G184* 2: N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 3: V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y; 4: E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 5: E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 6: K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

(relative to SEQ ID NO: 2 herein):

1: R181*,G182*,D183*,G184* 2: N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 3: V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y; 4: E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 5: E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 6: K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

(Relative to SEQ ID NO: 3 herein):

1: R179*,G180,I181*,G182* 2: N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 3: L204A,R,D,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V; 4: E210A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 5: E214A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 6: S267A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V

Relative to SEQ ID NO: 4 herein):

1: Q178*,G179* 2: N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 3: I201A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V; 4: D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 5: E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 6: Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

(relative to SEQ ID NO: 5 herein):

1: R176*,G177*,E178,G179* 2: N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 3: V201A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y; 4: D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 5: E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 6: Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

(relative to SEQ ID NO: 6 herein):

1: R181*,G182*,H183*,G184* 2: N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 3: I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V; 4: E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 5: E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 6: K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V; and

(relative to SEQ ID NO: 24)

1: R181*,G182*,H183*,G184* 2: N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 3: I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V; 4: E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 5: E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 6: K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V.

Contemplated according to the present invention is combining three, four, five or six mutation.

Specific double mutations for backbone SEQ ID NO: 1-6 and SEQ ID NO: 24 and SEQ ID NO: 26 are listed in the following.

Using SEQ ID NO: 1 as the backbone the following double mutations are contemplated according to the invention:

R181*/G182*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G182*/T183*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

T183*/G184*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R181*/G182*/V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

G182*/T183*/V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

T183*/G184*/V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

R181*/G182*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G182*/T183*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

T183*/G184*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V:

R181*/G182*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G182*/T183*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

T183*/G184*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R181*/G182*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

G182*/T183*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

T183*/G184*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V /V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V /E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V /E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V /K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y /E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y /E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y /K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V /E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V /K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V /K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

Using SEQ ID NO: 2 as the backbone the following double mutations are contemplated according to the invention:

R181*/G182*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G182*/D183*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

D183*/G184*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R181*/G182*/V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

G182*/T183*/V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

T183*/G184*/V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

R181*/G182*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G182*/T183*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

T183*/G184*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R181*/G182*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G182*/T183*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

T183*/G184*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R181*/G182*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

G182*/T183*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

T183*/G184*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y

/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

Using SEQ ID NO: 3 as the backbone the following double mutations are contemplated according to the invention:

R179*/G180*/N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G180*/I181*/N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

I181*/G182*/N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R179*/G180*/L204A,R,D,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;

G180*/I181*/L204A,R,D,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;

I181*/G182*/L204A,R,D,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;

R179*/G180*/E210A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G180*/I181*/E210A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

I181*/G182*/E210A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R179*/G180*/E214A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G180*/I181*/E214A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

I181*/G182*/E214A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R179*/G180*/S267A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

G180*/I181*/S267A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

I181*/G182*/S267A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/L204A,R,D,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;

N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E210A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E214A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/S267A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

L204A,R,D,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V

/E210A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

L204A,R,D,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V

/E214A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

L204A,R,D,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V

/S267A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

E210A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

E210A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/S267A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

E214A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/S267A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

Using SEQ ID NO: 4 as the backbone the following double mutations are contemplated according to the invention:

Q178*/G179*/N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Q178*/G179*/I201A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

Q178*/G179*/D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Q178*/G179*/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R179*/G180*/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N190/I201A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

N190/D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N190/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N190/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

I201/D207A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

I201/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

I201/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

D207/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

D207/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

E211/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

Using SEQ ID NO: 5 as the backbone the following double mutations are contemplated according to the invention:

R176*/G177*/N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G177*/E178*/N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

E178*/G179*/N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R176*/G177*/V201A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

G176*/E178*/V201A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

E178*/G179*/V201A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

R176*/G177*/D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G177*/E178*/D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

E178*/G179*/D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R176*/G177*/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G177*/E178*/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

E178*/G179*/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R176*/G177*/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G177*/E178*/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

E178*/G179*/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/V201A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;

N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

V201A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y

/D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

V201A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y

/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

V201A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y

/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.

Using SEQ ID NO: 6 as the backbone the following double mutations are contemplated according to the invention:

R181*/G182*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G182*/H183*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

H183*/G184*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R181*/G182*/I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

G182*/H183*/I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

H183*/G184*/I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

R181*/G182*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G182*/H183*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

H183*/G184*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R181*/G182*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G182*/H183*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

H183*/G184*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R181*/G182*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

G182*/H183*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

H183*/G184*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V

/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V /E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V /K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V /K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V.

Using SEQ ID NO: 24 as the backbone the following double mutations are contemplated according to the invention:

R181*/G182*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G182*/H183*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

H183*/G184*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R181*/G182*/I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

G182*/H183*/I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

H183*/G184*/I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

R181*/G182*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G182*/H183*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

H183*/G184*/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R181*/G182*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

G182*/H183*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

H183*/G184*/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

R181*/G182*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

G182*/H183*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

H183*/G184*/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V

/E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V /E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V /K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V /K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V.

All Termamyl-like alpha-amylase defined above may suitably be used as backbone for preparing variants of the invention.

However, in a preferred embodiment the variant comprises the following mutations: N190F/Q264S in SEQ ID NO: 4 or in corresponding positions in another parent Termamyl-like alpha-amylases.

In another embodiment the variant of the invention comprises the following mutations: I181*/G182*/N193F in SEQ ID NO: 3 (TVB146) or in corresponding positions in another parent Termamyl-like alpha-amylases. Said variant may further comprise a substitution in position E214Q.

In a preferred embodiment of the invention the parent Termamyl-like alpha-amylase is a hybrid alpha-amylase of SEQ ID NO: 4 and SEQ ID NO: 5. Specifically, the parent hybrid Termamyl-like alpha-amylase may be a hybrid alpha-amylase comprising the 445 C-terminal amino acid residues of the B. licheniformis alpha-amylase shown in SEQ ID NO: 4 and the 37 N-terminal amino acid residues of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 5, which may suitably further have the following mutations: H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 4). The latter mentioned hybrid is used in the examples below and is referred to as LE174.

General Mutations of the Invention

It may be preferred that a variant of the invention comprises one or more modifications in addition to those outlined above. Thus, it may be advantageous that one or more proline residues present in the part of the alpha-amylase variant which is modified is/are replaced with a non-proline residue which may be any of the possible, naturally occurring non-proline residues, and which preferably is an alanine, glycine, serine, threonine, valine or leucine.

Analogously, it may be preferred that one or more cysteine residues present among the amino acid residues with which the parent alpha-amylase is modified is/are replaced with a non-cysteine residue such as serine, alanine, threonine, glycine, valine or leucine.

Furthermore, a variant of the invention may—either as the only modification or in combination with any of the above outlined modifications—be modified so that one or more Asp and/or Glu present in an amino acid fragment corresponding to the amino acid fragment 185-209 of SEQ ID NO: 4 is replaced by an Asn and/or Gln, respectively. Also of interest is the replacement, in the Termamyl-like alpha-amylase, of one or more of the Lys residues present in an amino acid fragment corresponding to the amino acid fragment 185-209 of SEQ ID NO: 4 by an Arg.

It will be understood that the present invention encompasses variants incorporating two or more of the above outlined modifications.

Furthermore, it may be advantageous to introduce point-mutations in any of the variants described herein.

Mutations with may suitably made include mutations in the following positions: H133, M15, M197, A209.

Cloning a DNA Sequence Encoding an Alpha-Amylase

The DNA sequence encoding a parent alpha-amylase may be isolated from any cell or microorganism producing the alpha-amylase in question, using various methods well known in the art. First, a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the alpha-amylase to be studied. Then, if the amino acid sequence of the alpha-amylase is known, homologous, labelled oligonucleotide probes may be synthesized and used to identify alpha-amylase-encoding clones from a genomic library prepared from the organism in question. Alternatively, a labelled oligonucleotide probe containing sequences homologous to a known alpha-amylase gene could be used as a probe to identify alpha-amylase-encoding clones, using hybridization and washing conditions of lower stringency.

Yet another method for identifying alpha-amylase-encoding clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming alpha-amylase-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for alpha-amylase, thereby allowing clones expressing the alpha-amylase to be identified.

Alternatively, the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g., the phosphoroamidite method described by Beaucage and Caruthers (1981) or the method described by Matthes et al. (1984). In the phosphoroamidite method, oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, the fragments corresponding to various parts of the entire DNA sequence), in accordance with standard techniques. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or Saiki et al. (1988).

Site-Directed Mutagenesis

Once an alpha-amylase-encoding DNA sequence has been isolated, and desirable sites for mutation identified, mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis. In a specific method, a single-stranded gap of DNA, bridging the alpha-amylase-encoding sequence, is created in a vector carrying the alpha-amylase gene. Then the synthetic nucleotide, bearing the desired mutation, is annealed to a homologous portion of the single-stranded DNA. The remaining gap is then filled in with DNA polymerase I (Klenow fragment) and the construct is ligated using T4 ligase. A specific example of this method is described in Morinaga et al. (1984). U.S. Pat. No. 4,760,025 discloses the introduction of oligonucleotides encoding multiple mutations by performing minor alterations of the cassette. However, an even greater variety of mutations can be introduced at any one time by the Morinaga method, because a multitude of oligonucleotides, of various lengths, can be introduced.

Another method for introducing mutations into alpha-amylase-encoding DNA sequences is described in Nelson and Long (1989). It involves the 3-step generation of a PCR fragment containing the desired mutation introduced by using a chemically synthesized DNA strand as one of the primers in the PCR reactions. From the PCR-generated fragment, a DNA fragment carrying the mutation may be isolated by cleavage with restriction endonucleases and reinserted into an expression plasmid.

Random Mutagenesis

Random mutagenesis is suitably performed either as localised or region-specific random mutagenesis in at least three parts of the gene translating to the amino acid sequence shown in question, or within the whole gene.

The random mutagenesis of a DNA sequence encoding a parent alpha-amylase may be conveniently performed by use of any method known in the art.

In relation to the above, a further aspect of the present invention relates to a method for generating a variant of a parent alpha-amylase, e.g., wherein the variant exhibits altered or increased thermal stability relative to the parent, the method comprising:

(a) subjecting a DNA sequence encoding the parent alpha-amylase to random mutagenesis,

(b) expressing the mutated DNA sequence obtained in step (a) in a host cell, and

(c) screening for host cells expressing analpha-amylase variant which has an altered property (i.e., thermal stability) relative to the parent alpha-amylase.

Step (a) of the above method of the invention is preferably performed using doped primers.

For instance, the random mutagenesis may be performed by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the random mutagenesis may be performed by use of any combination of these mutagenizing agents. The mutagenizing agent may, e.g., be one which induces transitions, transversions, inversions, scrambling, deletions, and/or insertions.

Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) ir-radiation, hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues. When such agents are used, the mutagenesis is typically performed by incubating the DNA sequence encoding the parent enzyme to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions for the mutagenesis to take place, and selecting for mutated DNA having the desired properties.

When the mutagenesis is performed by the use of an oligonucleotide, the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the oligonucleotide at the positions which are to be changed. The doping or spiking may be done so that codons for unwanted amino acids are avoided. The doped or spiked oligonucleotide can be incorporated into the DNA encoding the alpha-amylase enzyme by any published technique, using e.g., PCR, LCR or any DNA polymerase and ligase as deemed appropriate.

Preferably, the doping is carried out using “constant random doping”, in which the percentage of wild-type and mutation in each position is predefined. Furthermore, the doping may be directed toward a preference for the introduction of certain nucleotides, and thereby a preference for the introduction of one or more specific amino acid residues. The doping may be made, e.g., so as to allow for the introduction of 90% wild type and 10% mutations in each position. An additional consideration in the choice of a doping scheme is based on genetic as well as protein-structural constraints. The doping scheme may be made by using the DOPE program which, inter alfa, ensures that introduction of stop codons is avoided.

When PCR-generated mutagenesis is used, either a chemically treated or non-treated gene encoding a parent alpha-amylase is subjected to PCR under conditions that increase the mis-incorporation of nucleotides (Deshler 1992; Leung et al., Technique, Vol. 1, 1989, pp. 11-15).

A mutator strain of E. coli (Fowler et al., 1974, Molec. Gen. Genet. 133: 179-191), S. cereviseae or any other microbial organism may be used for the random mutagenesis of the DNA encoding the alpha-amylase by, e.g., transforming a plasmid containing the parent glycosylase into the mutator strain, growing the mutator strain with the plasmid and isolating the mutated plasmid from the mutator strain. The mutated plasmid may be subsequently transformed into the expression organism.

The DNA sequence to be mutagenized may be conveniently present in a genomic or cDNA library prepared from an organism expressing the parent alpha-amylase. Alternatively, the DNA sequence may be present on a suitable vector such as a plasmid or a bacteriophage, which as such may be incubated with or other-wise exposed to the mutagenizing agent. The DNA to be mutagenized may also be present in a host cell either by being integrated in the genome of said cell or by being present on a vector harboured in the cell. Finally, the DNA to be mutagenized may be in isolated form. It will be understood that the DNA sequence to be subjected to random mutagenesis is preferably a cDNA or a genomic DNA sequence.

In some cases it may be convenient to amplify the mutated DNA sequence prior to performing the expression step b) or the screening step c). Such amplification may be performed in accordance with methods known in the art, the presently preferred method being PCR-generated amplification using oligonucleotide primers prepared on the basis of the DNA or amino acid sequence of the parent enzyme.

Subsequent to the incubation with or exposure to the mutagenizing agent, the mutated DNA is expressed by culturing a suitable host cell carrying the DNA sequence under conditions allowing expression to take place. The host cell used for this purpose may be one which has been transformed with the mutated DNA sequence, optionally present on a vector, or one which was carried the DNA sequence encoding the parent enzyme during the mutagenesis treatment. Examples of suitable host cells are the following: gram positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, Streptomyces lividans or Streptomyces murinus; and gram-negative bacteria such as E. coli.

The mutated DNA sequence may further comprise a DNA sequence encoding functions permitting expression of the mutated DNA sequence.

Localized Random Mutagenesis

The random mutagenesis may be advantageously localized to a part of the parent alpha-amylase in question. This may, e.g., be advantageous when certain regions of the enzyme have been identified to be of particular importance for a given property of the enzyme, and when modified are expected to result in a variant having improved properties. Such regions may normally be identified when the tertiary structure of the parent enzyme has been elucidated and related to the function of the enzyme.

The localized, or region-specific, random mutagenesis is conveniently performed by use of PCR generated mutagenesis techniques as described above or any other suitable technique known in the art. Alternatively, the DNA sequence encoding the part of the DNA sequence to be modified may be isolated, e.g., by insertion into a suitable vector, and said part may be subsequently subjected to mutagenesis by use of any of the mutagenesis methods discussed above.

Alternative Methods of Providing Alpha-Amylase Variants

Alternative methods for providing variants of the invention include gene shuffling method known in the art including the methods, e.g., described in WO 95/22625 (from Affymax Technologies N.V.) and WO 96/00343 (from Novo Nordisk A/S).

Expression of Alpha-Amylase Variants

According to the invention, a DNA sequence encoding the variant produced by methods described above, or by any alternative methods known in the art, can be expressed, in enzyme form, using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.

The recombinant expression vector carrying the DNA sequence encoding an alpha-amylase variant of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

In the vector, the DNA sequence should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA sequence encoding an alpha-amylase variant of the invention, especially in a bacterial host, are the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis alpha-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens alpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.

The expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably connected to the DNA sequence encoding the alpha-amylase variant of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.

The vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B. licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, e.g., as described in WO 91/17243.

While intracellular expression may be advantageous in some respects, e.g., when using certain bacteria as host cells, it is generally preferred that the expression is extracellular. In general, the Bacillus alpha-amylases mentioned herein comprise a preregion permitting secretion of the expressed protease into the culture medium. If desirable, this preregion may be replaced by a different preregion or signal sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective preregions.

The procedures used to ligate the DNA construct of the invention encoding an alpha-amylase variant, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989).

The cell of the invention, either comprising a DNA construct or an expression vector of the invention as defined above, is advantageously used as a host cell in the recombinant production of an alpha-amylase variant of the invention. The cell may be transformed with the DNA construct of the invention encoding the variant, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.

The cell of the invention may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g., a bacterial or a fungal (including yeast) cell.

Examples of suitable bacteria are gram-positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyces lividans or Streptomyces murinus, or gram-negative bacteria such as E. coli. The transformation of the bacteria may, for instance, be effected by protoplast transformation or by using competent cells in a manner known per se.

The yeast organism may favourably be selected from a species of Saccharomyces or Schizosaccharomyces, e.g., Saccharomyces cerevisiae. The filamentous fungus may advantageously belong to a species of Aspergillus, e.g., Aspergillus oryzae or Aspergillus niger. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.

In yet a further aspect, the present invention relates to a method of producing an alpha-amylase variant of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the variant and recovering the variant from the cells and/or culture medium.

The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the alpha-amylase variant of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).

The alpha-amylase variant secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

Compositions

In a still further aspect, the present invention relates to compositions comprising an alpha-amylase or alpha-amylase variant of the present invention. Preferably, the compositions are enriched in an alpha-amylase or alpha-amylase variant of the present invention. In the present context, the term “enriched” indicates that the alpha-amylase activity of the composition has been increased, e.g., with an enrichment factor of 1.1.

The composition may comprise an alpha-amylase or alpha-amylase variant of the invention as the major enzymatic component, e.g., a mono-component composition. Alternatively, the composition may comprise multiple enzymatic activities, such as an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase. The additional enzyme(s) may be producible by means of a microorganism belonging to the genus Aspergillus, preferably Aspergillus aculeatus, Aspergillus awamori, Aspergillus niger, or Aspergillus oryzae, or Trichoderma, Humicola, preferably Humicola insolens, or Fusarium, preferably Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, or Fusarium venenatum.

The alpha-amylase compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. For instance, the alpha-amylase composition may be in the form of a granulate or a microgranulate. The polypeptide to be included in the composition may be stabilized in accordance with methods known in the art.

Examples are given below of preferred uses of the alpha-amylase compositions of the invention. The dosage of the alpha-amylase composition of the invention and other conditions under which the composition is used may be determined on the basis of methods known in the art.

Further Compositions

The invention also relates to a composition comprising a mixture of one or more alpha-amylase or alpha-amylase variant of the invention derived from (as the parent Termamyl-like alpha-amylase) the B. stearothermophilus alpha-amylase having the sequence shown in SEQ ID NO: 3 and a Termamyl-like alpha-amylase derived from the B. licheniformis alpha-amylase having the sequence shown in SEQ ID NO: 4.

Further, the invention also relates to a composition comprising a mixture of one or more variants according the invention derived from (as the parent Termamyl-like alpha-amylase) the B. stearothermophilus alpha-amylase having the sequence shown in SEQ ID NO: 3 and a hybrid alpha-amylase comprising a part of the B. amyloliquefaciens alpha-amylase shown in SEQ ID NO: 5 and a part of the B. licheniformis alpha-amylase shown in SEQ ID NO: 4. The latter mentioned hydrid Termamyl-like alpha-amylase comprises the 445 C-terminal amino acid residues of the B. licheniformis alpha-amylase shown in SEQ ID NO: 4 and the 37 N-terminal amino acid residues of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 5. Said latter mentioned hybrid alpha-amylase may suitably comprise the following mutations: H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 4). In the examples below said hybrid parent Termamyl-like alpha-amylase, is used in combination with variants of the invention, which variants may be used in compositions of the invention.

In a specific embodiment of the invention the composition comprises a mixture of TVB146 and LE174, e.g., in a ratio of 2:1 to 1:2, such as 1:1.

An alpha-amylase or alpha-amylase variant of the invention or a composition of the invention may in an aspect of the invention be used for washing and/or dishwashing; for textile desizing or for starch liquefaction.

Detergent Compositions

The alpha-amylase or alpha-amylase variant of the invention may be added to and thus become a component of a detergent composition.

The detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent additive comprising the enzyme of the invention. The detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase.

In general the properties of the chosen enzyme(s) should be compatible with the selected detergent, (i.e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.

Proteases: Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. The protease may be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g., of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.

Preferred commercially available protease enzymes include Alcalase®, Savinase®, Primase®, Duralase®, Esperase®, and Kannase® (Novo Nordisk A/S), Maxatase®, Maxacal, Maxapem®, Properase®, Purafect®, Purafect OxP®, FN2®, and FN3® (Genencor International Inc.).

Lipases: Suitable lipases include those of bac-terial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g., from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g., from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).

Other examples are lipase variants such as those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.

Preferred commercially available lipase enzymes include Lipolase™ and Lipolase Ultra™ (Novo Nordisk A/S).

Amylases: Suitable amylases (alpha- and/or beta-) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g., a special strain of B. licheniformis, described in more detail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ and BAN™ (Novo Nordisk A/S), Rapidase™ and Purastar™ (from Genencor Interna-tional Inc.).

Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Commercially available cellulases include Celluzyme®, and Carezyme® (Novo Nordisk A/S), Clazinase®, and Puradax HA® (Genencor International Inc.), and KAC-500(B)® (Kao Corporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g., from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme® (Novo Nordisk A/S).

The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, i.e., a separate additive or a combined additive, can be formulated e.g., as a granulate, a liquid, a slurry, etc. Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonyl-phenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic. The surfactants are typically present at a level of from 0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.

When included therein the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonyl-phenol ethoxylate, alkylpolyglycoside, alkyldimethylamine-oxide, ethoxylated fatty acid monoethanol-amide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexing agent such as zeolite, diphosphate, tripho-sphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetri-aminepen-taacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂ source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. Alternatively, the bleaching system may comprise peroxyacids of, e.g., the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the inven-tion may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the com-position may be formulated as described in, e.g., WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredients such as, e.g., fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions any enzyme, in particular the enzyme of the invention, may be added in an amount corresponding to 0.01-100 mg of enzyme protein per liter of wash liquor, preferably 0.05-5 mg of enzyme protein per liter of wash liquor, in particular 0.1-1 mg of enzyme protein per liter of wash liquor.

The enzyme of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202 which is hereby incorporated as reference.

Further Detergent Compositions

As mentioned above, variants of the invention may suitably be incorporated in detergent compositions. Increased thermostability at low calcium concentrations would be very beneficial for amylase performance in detergents, i.e., the alkaline region. Reference is made, for example, to WO 96/23874 and WO 97/07202 for further details concerning relevant ingredients of detergent compositions (such as laundry or dishwashing detergents), appropriate methods of formulating the variants in such detergent compositions, and for examples of relevant types of detergent compositions.

Detergent compositions comprising an alpha-amylase or alpha-amylase variant of the invention may additionally comprise one or more other enzymes, such as a lipase, cutinase, protease, cellulase, peroxidase or laccase, and/or another alpha-amylase.

An alpha-amylase or alpha-amylase variant of the invention may be incorporated in detergents at conventionally employed concentrations. It is at present contemplated that a variant of the invention may be incorporated in an amount corresponding to 0.00001-1 mg (calculated as pure, active enzyme protein) of alpha-amylase per liter of wash/dishwash liquor using conventional dosing levels of detergent.

Dishwash Deterget Compositions

The alpha-amylase or alpha-amylase variant of the invention may also be used in dishwash detergent compositions, including the following:

1) Powder Automatic Dishwashing Composition

Nonionic surfactant 0.4-2.5%  Sodium metasilicate 0-20% Sodium disilicate 3-20% Sodium triphosphate 20-40%  Sodium carbonate 0-20% Sodium perborate  2-9% Tetraacetyl ethylene diamine (TAED)  1-4% Sodium sulphate 5-33% Enzymes 0.0001-0.1%    

21 Powder Automatic Dishwashing Composition

Nonionic surfactant  1-2% (e.g., alcohol ethoxylate) Sodium disilicate 2-30% Sodium carbonate 10-50%  Sodium phosphonate  0-5% Trisodium citrate dihydrate 9-30% Nitrilotrisodium acetate (NTA) 0-20% Sodium perborate monohydrate 5-10% Tetraacetyl ethylene diamine (TAED)  1-2% Polyacrylate polymer 6-25% (e.g., maleic acid/acrylic acid copolymer) Enzymes 0.0001-0.1%     Perfume 0.1-0.5%  Water 5-10

3) Powder Automatic Dishwashing Composition

Nonionic surfactant 0.5-2.0%  Sodium disilicate 25-40%  Sodium citrate 30-55%  Sodium carbonate 0-29% Sodium bicarbonate 0-20% Sodium perborate monohydrate 0-15% Tetraacetyl ethylene diamine (TAED)  0-6% Maleic acid/acrylic acid copolymer  0-5% Clay  1-3% Polyamino acids 0-20% Sodium polyacrylate  0-8% Enzymes 0.0001-0.1%    

4) Powder Automatic Dishwashing Composition

Nonionic surfactant 1-2% Zeolite MAP 15-42%  Sodium disilicate 30-34%  Sodium citrate 0-12%  Sodium carbonate 0-20%  Sodium perborate monohydrate 7-15%  Tetraacetyl ethylene diamine (TAED) 0-3% Polymer 0-4% Maleic acid/acrylic acid copolymer 0-5% Organic phosphonate 0-4% Clay 1-2% Enzymes 0.0001-0.1%    Sodium sulphate Balance

5) Powder Automatic Dishwashing Composition

Nonionic surfactant  1-7% Sodium disilicate 18-30% Trisodium citrate 10-24% Sodium carbonate 12-20% Monopersulphate (2 KHSO₅•KHSO₄•K₂SO₄) 15-21% Bleach stabilizer  0.1-2% Maleic acid/acrylic acid copolymer  0-6% Diethylene triamine pentaacetate, pentasodium salt  0-2.5% Enzymes 0.0001-0.1%   Sodium sulphate, water Balance 6) Powder and Liquid Dishwashing Composition with Cleaning Surfactant System

Nonionic surfactant 0-1.5%  Octadecyl dimethylamine N-oxide dihydrate 0-5% 80:20 wt. C18/C16 blend of octadecyl 0-4% dimethylamine N-oxide dihydrate and hexadecyldimethyl amine N-oxide dihydrate 70:30 wt. C18/C16 blend of octadecyl bis 0-5% (hydroxyethyl)amine N-oxide anhydrous and hexadecyl bis (hydroxyethyl)amine N-oxide anhydrous C₁₃-C₁₅ alkyl ethoxysulfate with an average degree 0-10%  of ethoxylation of 3 C₁₂-C₁₅ alkyl ethoxysulfate with an average degree 0-5% of ethoxylation of 3 C₁₃-C₁₅ ethoxylated alcohol with an average degree 0-5% of ethoxylation of 12 A blend of C₁₂-C₁₅ ethoxylated alcohols with an 0-6.5%  average degree of ethoxylation of 9 A blend of C₁₃-C₁₅ ethoxylated alcohols with an 0-4% average degree of ethoxylation of 30 Sodium disilicate 0-33%  Sodium tripolyphosphate 0-46%  Sodium citrate 0-28%  Citric acid 0-29%  Sodium carbonate 0-20%  Sodium perborate monohydrate 0-11.5%   Tetraacetyl ethylene diamine (TAED) 0-4% Maleic acid/acrylic acid copolymer 0-7.5%  Sodium sulphate 0-12.5%   Enzymes 0.0001-0.1%   

7) Non-Aqueous Liquid Automatic Dishwashing Composition

Liquid nonionic surfactant  2.0-10.0% (e.g., alcohol ethoxylates) Alkali metal silicate  3.0-15.0% Alkali metal phosphate 20.0-40.0% Liquid carrier selected from higher 25.0-45.0% glycols, polyglycols, polyoxides, glycolethers Stabilizer  0.5-7.0% (e.g., a partial ester of phosphoric acid and a C₁₆-C₁₈ alkanol) Foam suppressor (e.g., silicone)    0-1.5% Enzymes 0.0001-0.1% 

8) Non-Aqueous Liquid Dishwashing Composition

Liquid nonionic surfactant 2.0-10.0% (e.g., alcohol ethoxylates) Sodium silicate 3.0-15.0% Alkali metal carbonate 7.0-20.0% Sodium citrate  0.0-1.5% Stabilizing system  0.5-7.0% (e.g., mixtures of finely divided silicone and low molecular weight dialkyl polyglycol ethers) Low molecule weight polyacrylate polymer 5.0-15.0% Clay gel thickener (e.g., bentonite) 0.0-10.0% Hydroxypropyl cellulose polymer  0.0-0.6% Enzymes 0.0001-0.1%  Liquid carrier selected from higher lycols, Balance polyglycols, polyoxides and glycol ethers

9) Thixotropic Liquid Automatic Dishwashing Composition

C₁₂-C₁₄ fatty acid 0-0.5% Block co-polymer surfactant 1.5-15.0%   Sodium citrate  0-12% Sodium tripolyphosphate  0-15% Sodium carbonate  0-8% Aluminium tristearate 0-0.1% Sodium cumene sulphonate 0-1.7% Polyacrylate thickener 1.32-2.5%   Sodium polyacrylate 2.4-6.0%  Boric acid 0-4.0% Sodium formate 0-0.45%  Calcium formate 0-0.2% Sodium n-decydiphenyl oxide disulphonate 0-4.0% Monoethanol amine (MEA) 0-1.86%  Sodium hydroxide (50%) 1.9-9.3%  1,2-Propanediol 0-9.4% Enzymes 0.0001-0.1%    Suds suppressor, dye, perfumes, water Balance

10) Liquid Automatic Dishwashing Composition

Alcohol ethoxylate 0-20% Fatty acid ester sulphonate 0-30% Sodium dodecyl sulphate 0-20% Alkyl polyglycoside 0-21% Oleic acid 0-10% Sodium disilicate monohydrate 18-33%  Sodium citrate dihydrate 18-33%  Sodium stearate 0-2.5%  Sodium perborate monohydrate 0-13% Tetraacetyl ethylene diamine (TAED)  0-8% Maleic acid/acrylic acid copolymer  4-8% Enzymes 0.0001-0.1%    

11) Liquid Automatic Dishwashing Composition Containing Protected Bleach Particles

Sodium silicate 5-10%  Tetrapotassium pyrophosphate 15-25%  Sodium triphosphate 0-2% Potassium carbonate 4-8% Protected bleach particles, e.g., chlorine 5-10%  Polymeric thickener 0.7-1.5%    Potassium hydroxide 0-2% Enzymes 0.0001-0.1%    Water Balance 11) Automatic dishwashing compositions as described in 1), 2), 3), 4), 6) and 10), wherein perborate is replaced by percarbonate. 12) Automatic dishwashing compositions as described in 1)-6) which additionally contain a manganese catalyst. The manganese catalyst may, e.g., be one of the compounds described in “Efficient manganese catalysts for low-temperature bleaching”, Nature 369: 637-639 (1994).

Uses

The present invention is also directed to methods for using an alpha-amylase or alpha-amylase variant of the invention in detergents, in particular laundry detergent compositions and dishwash detergent compositions.

INDUSTRIAL APPLICATIONS

An alpha-amylase and alpha-amylase variant of the invention are well suited for use in a variety of industrial processes, in particular the enzymes of the invention finds potential applications as a component in detergents, e.g., laundry, dishwash and hard surface cleaning detergent compositions, but it may also be useful in the production of sweeteners and ethanol from starch. Thus, it may be used in conventional starch-converting processes, such as liquefaction and saccharification processes described in U.S. Pat. No. 3,912,590 and EP patent publications Nos. 252,730 and 63,909.

An alpha-amylase or alpha-amylase variant of the invention may also be used in the production of lignocellulosic materials, such as pulp, paper and cardboard, from starch reinforced waste paper and cardboard, especially where repulping occurs at pH above 7 and where amylases can facilitate the disintegration of the waste material through degradation of the reinforcing starch. The alpha-amylase of the invention is especially useful in a process for producing a papermaking pulp from starch-coated printed paper. The process may be performed as described in WO 95/14807, comprising the following steps:

a) disintegrating the paper to produce a pulp,

b) treating with a starch-degrading enzyme before, during or after step a), and

c) separating ink particles from the pulp after steps a) and b).

An alpha-amylase or alpha-amylase variant of the invention may also be very useful in modifying starch where enzymatically modified starch is used in papermaking together with alkaline fillers such as calcium carbonate, kaolin and clays. With the alkaline alpha-amylases of the invention it becomes possible to modify the starch in the presence of the filler thus allowing for a simpler integrated process.

An alpha-amylase or alpha-amylase variant of the invention may also be very useful in textile desizing. In the textile processing industry, alpha-amylases are traditionally used as auxiliaries in the desizing process to facilitate the removal of starch-containing size which has served as a protective coating on weft yarns during weaving. Complete removal of the size coating after weaving is import-ant to ensure optimum results in the subsequent processes, in which the fabric is scoured, bleached and dyed. Enzymatic starch break-down is preferred because it does not involve any harmful effect on the fiber material. In order to reduce processing cost and increase mill through-put, the desizing processing is sometimes combined with the scouring and bleaching steps. In such cases, non-enzymatic auxiliaries such as alkali or oxidation agents are typically used to break down the starch, because traditional alpha-amylases are not very compatible with high pH levels and bleaching agents. The non-enzymatic breakdown of the starch size does lead to some fiber damage because of the rather aggressive chemicals used. Accordingly, it would be desirable to use the alpha-amylases of the invention as they have an improved performance in alkaline solutions. The alpha-amylases may be used alone or in combination with a cellulase when desizing cellulose-containing fabric or textile.

The alpha-amylases of the invention may also be very useful in a beer-making process; the alpha-amylases will typically be added during the mashing process.

Production of Sweeteners from Starch

A “traditional” process for conversion of starch to fructose syrups normally consists of three consecutive enzymatic processes, viz., a liquefaction process followed by a saccharification process and an isomerization process. During the liquefaction process, starch is degraded to dextrins by an alpha-amylase (e.g., Termamyl™) at pH values between 5.5 and 6.2 and at temperatures of 95-160° C. for a period of approx. 2 hours. In order to ensure an optimal enzyme stability under these conditions, 1 mM of calcium is added (40 ppm free calcium ions).

After the liquefaction process the dextrins are converted into dextrose by addition of a glucoamylase (e.g., AMG™) and a debranching enzyme, such as an isoamylase or a pullulanase (e.g., Promozyme™). Before this step the pH is reduced to a value below 4.5, maintaining the high temperature (above 95° C.), and the liquefying alpha-amylase activity is denatured. The temperature is lowered to 60° C., and glucoamylase and debranching enzyme are added. The saccharification process proceeds for 24-72 hours.

After the saccharification process the pH is increased to a value in the range of 6-8, preferably pH 7.5, and the calcium is removed by ion exchange. The dextrose syrup is then converted into high fructose syrup using, e.g., an immmobilized glucoseisomerase (such as Sweetzyme™)

At least 1 enzymatic improvements of this process could be envisaged.

Reduction of the calcium dependency of the liquefying alpha-amylase. Addition of free calcium is required to ensure adequately high stability of the alpha-amylase, but free calcium strongly inhibits the activity of the glucoseisomerase and needs to be removed, by means of an expensive unit operation, to an extent which reduces the level of free calcium to below 3-5 ppm. Cost savings could be obtained if such an operation could be avoided and the liquefaction process could be performed without addition of free calcium ions.

To achieve that, a less calcium-dependent Termamyl-like alpha-amylase which is stable and highly active at low concentrations of free calcium (<40 ppm) is required. Such a Termamyl-like alpha-amylase should have a pH optimum at a pH in the range of 4.5-6.5, preferably in the range of 4.5-5.5.

Materials and Methods

Chemicals used as buffers and substrates were commercial products of at least reagent grade.

Enzymes:

SP690: alpha-amylase shown in SEQ ID NO: 1 SP722: alpha-amylase shown in SEQ ID NO: 2 Termamyl®: alpha-amylase from Bacillus licheniformis shown in SEQ ID NO: 4. AA560: alpha-amylase of the invention shown in SEQ ID NO: 24 encoded by the DNA sequence shown in SEQ ID NO: 23. AA360: alpha-amylase shown in SEQ ID NO: 26 being identical to the AA560 alpha-amylase encoded by the DNA sequence shown in SEQ ID NO: 25. BSG alpha-amylase: B. stearothermophilus alpha-amylase depicted in SEQ ID NO: 3. TVB146 alpha-amylase variant: B. stearothermophilus alpha-amylase variant depicted in SEQ ID NO: 3 with the following mutations: with the deletion in positions 1181-G182+N193F. LE174 hybrid alpha-amylase variant: LE174 is a hybrid Termamyl-like alpha-amylase being identical to the Termamyl sequence, i.e., the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 4, except that the N-terminal 35 amino acid residues (of the mature protein) has been replaced by the N-terminal 33 residues of BAN (mature protein), i.e., the Bacillus amyloliquefaciens alpha-amylase shown in SEQ ID NO: 5, which further have the following mutations: H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 4). LE174 was constructed by SOE-PCR (Higuchi et al., 1988, Nucleic Acids Research 16:7351).

Model Detergent:

A/P (Asia/Pacific) Model Detergent has the following composition: 20% STPP (sodium tripolyphosphate), 25% Na₂SO₄, 15% Na₂CO₃, 20% LAS (linear alkylbenzene sulfonate, Nansa 80S), 5% C₁₂-C₁₅ alcohol ethoxylate (Dobanol 25-7), 5% Na₂Si₂O₅, 0.3% NaCl.

Omo Multi Acao (Brazil),

Omo concentrated powder (Europe) (product of Unilever) Ariel Futur liquid (Europe) (product of Procter and Gamble)

Deposit of Biological Material

The following biological material has been deposited under the terms of the Budapest Treaty with the Deutshe Sammmlung von Microorganismen and Zellkulturen GmbH (DSMZ), Mascheroder Weg 1 b, D-38124 Braunschweig DE, and given the following accession number:

Deposit Accession Number Date of Deposit NN017557 DSM 12648 25 Jan. 1999 NN017560 DSM 12649 25 Jan. 1999 NN049467 DSM12761 7^(th) Apr. 1999 NN049470 DSM12764 7^(th) Apr. 1999

The strains have been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposit represents a substantially pure culture of the deposited strain. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Host Organism

Bacillus subtilis strain SHa273 is disclosed in WO 95/10603 E. coli strain SJ2 (Diderichsen et al. (1990)).

Plasmids:

The gene bank vector pSJ1678 is disclosed in WO 94/19454 which is hereby incorporated by reference.

pTVB110 is a plasmid replicating in Bacillus subtilis by the use of origin of replication from pUB110 (Gryczan, 1978, J. Bact. 134:318-329). The plasmid further encodes the cat gene, conferring resistance towards chlorampenicol, obtained from plasmid pC194 (Horinouchi and Weisblum, 1982, J. Bact. 150: 815-825). The plasmid harbors a truncated version of the Bacillus licheniformis alpha-amylase gene, amyL, such that the amyL promoter, signal sequence and transcription terminator are present, but the plasmid does not provide an amy-plus phenotype (halo formation on starch containing agar).

Methods General Molecular Biology Methods:

Unless otherwise mentioned the DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al. (1989); Ausubel et al. (1995); Harwood and Cutting (1990).

Fermentation and Purification of Alpha-Amylase Variants

Fermentation may be performed by methods well known in the art or as follows.

A B. subtilis strain harbouring the relevant expression plasmid is streaked on an LB-agar plate with 10 μg/ml kanamycin from −80° C. stock, and grown overnight at 37° C.

The colonies are transferred to 100 ml BPX media supplemented with 10 μg/ml kanamycin in a 500 ml shaking flask.

Composition of BPX Medium:

Potato starch 100 g/l Barley flour 50 g/l BAN 5000 SKB 0.1 g/l Sodium caseinate 10 g/l Soy Bean Meal 20 g/l Na₂HPO₄, 12 H₂O 9 g/l Pluronic ™ 0.1 g/l

The culture is shaken at 37° C. at 270 rpm for 5 days.

Cells and cell debris are removed from the fermentation broth by centrifugation at 4500 rpm in 20-25 minutes. Afterwards the supernatant is filtered to obtain a completely clear solution. The filtrate is concentrated and washed on a UF-filter (10000 cut off membrane) and the buffer is changed to 20 mM Acetate pH 5.5. The UF-filtrate is applied on a S-sepharose F.F. and elution is carried out by step elution with 0.2M NaCl in the same buffer. The eluate is dialysed against 10 mM Tris, pH 9.0 and applied on a Q-sepharose F.F. and eluted with a linear gradient from 0-0.3 M NaCl over 6 column volumes. The fractions which contain the activity (measured by the Phadebas assay) are pooled, pH was adjusted to pH 7.5 and remaining color was removed by a treatment with 0.5% W/vol. active coal in 5 minutes.

Assays for Determining Alpha-Amylase Activity Activity Determination—(KNU)

One Kilo alpha-amylase Unit (1 KNU) is the amount of enzyme which breaks down 5.26 g starch (Merck, Amylum Solubile, Erg. B 6, Batch 9947275) per hour in Novo Nordisk's standard method for determination of alpha-amylase based upon the following condition:

Substrate soluble starch Calcium content in solvent 0.0043M Reaction time 7-20 minutes Temperature 37° C. pH 5.6

Detailed description of Novo Nordisk's analytical method (AF 9) is available on request.

BS-Amylase Activity Determination—KNU(S) 1. Application Field

This method is used to determine alpha-amylase activity in fermentation and recovery samples and formulated and granulated products.

2. Principle

BS-amylase breaks down the substrate (4,6-ethylidene(G₇)-p-nitrophenyl(G₁)-alpha,D-maltoheptaoside (written as ethylidene-G₇-PNP) into, among other things, G₂-PNP and G₃-PNP, where G denoted glucose and PNP p-nitrophenol.

G2-PNP and G3-PNP are broken down by alpha-glucosidase, which is added in excess, into glucose and the yellow-coloured p-nitrophenol.

The colour reaction is monitored in situ and the change in absorbance over time calculated as an expression of the spreed of the reaction and thus of the activity of the enzyme. See the Boehringer Mannheim 1442 309 guidelines for further details.

2.1 Reaction Conditions Reaction:

Temperature: 37° C.

pH: 7.1

Pre-incubation time: 2 minutes

Detection:

Wavelength: 405 nm

Measurement time: 3 minutes

3. Definition of Units

Bacillus stearothermophius alpha-amylase (BS-amylase) activity is determined relative to a standard of declared activity and stated in Kilo Novo Units (Stearothermophilus) or KNU(S)).

4. Specificity and Sensitivity

Limit of determination: approx. 0.4 KNU(s)/g

5. Apparatus

Cobas Fara analyser

Diluted (e.g., Hamilton Microlab 1000)

Analytical balance (e.g., Mettler AE 100) Stirrer plates

6. Reagents/Substrates

A ready-made kit is used in this analysis to determine alpha-amylase activity. Note that the reagents specified for the substrate and alpha-glucosidase are not used as described in the Boehringer Mannheim guidelines. However, the designations “buffer”, “glass 1”, glass 1a” and Glass 2″ are those referred to in those guidelines.

6.1. Substrate

4,6-ethylidene(G₇)-p-nitrophenyl(G₁)-alpha,D-maltoheptaoside (written as ethylidene-G₇-PNP), e.g., Boehringer Mannheim 1442 309

6.2 Alpha-Glucosidase Help Reagent

alpha-glucosidase, e.g., Boehringer Mannheim 1442 309

6.3 BRIJ 35 Solution

BRIJ 35 (30% W/V Sigma 430 AG-6) 1000 mL Demineralized water up to 2,000 mL

6.4 Stabiliser

Brij 35 solution 33 mL CaCl₂*2H₂O (Merck 2382) 882 g Demineralized water up to 2,000 mL

7. Samples and Standards 7.1 Standard Curve Example: Preparation of BS-Amylase Standard Curve

The relevant standard is diluted to 0.60 KNU(s)/mL as follows. A calculated quantity of standard is weighed out and added to 200 mL volumetric flask, which is filled to around the ⅔ mark with demineralized water. Stabiliser corresponding to 1% of the volume of the flask is added and the flask is filled to the mark with demineralized water.

A Hamilton Microlab 1000 is used to produce the dilutions shown below. Demineralized water with 1% stabiliser is used as the diluent.

Dilution No. Enzyme stock solution 1% stabilizer KNU(s)/mL 1 20 μL 580 μL 0.02 2 30 μL 570 μL 0.03 3 40 μL 560 μL 0.04 4 50 μL 550 μL 0.05 5 60 μL 540 μL 0.06

7.2 Level Control

A Novo Nordisk A/S BS amylase level control is included in all runs using the Cobas Fara. The control is diluted with 1% stabilizer so that the final dilution is within the range of the standard curve. All weights and dilutions are noted on the worklist

7.3 Sample Solutions Single Determination

Fermentation samples (not final samples) from production, all fermentation samples from pilot plants and storage stability samples are weighed out and analyzed once only. Double determination over 1 run: Process samples, final fermentation samples from production, samples from GLP studies and R&D samples are weighed out and analyzed twice. Double determinations over 2 runs: Finished product samples are weighed out and analyzed twice over two separate runs. Maximum concentration of samples in powder form: 5% Test samples are diluted with demineralized water with 1% stabilizer to approx. 0.037 KNU(S)/mL on the basis of their expected activity. The final dilution is made direct into the sample cup.

8. Procedure 8.1 Cobas Menu Program

The Cobas Menu Program is used to suggest the weight/dilutions of samples and level control to be used.

The samples are entered into the program with a unique identification code and a worklist is printed out

The samples and control are weighed out and diluted as stated on the worklist with hand-written weight data is inserted into the BS-amylase analysis logbook

The results are computerized automatically by the Cobas Fara as described in item 9 and printed out along with the standard curve.

Worklists and results printouts are inserted into the BS-amylase analysis logbook.

8.2 Cobas Fara Set-Up

The samples are placed in the sample rack

The five standards are placed in the calibration rack at position 1 to 5 (strongest standard at position 5), and control placed in the same rack at position 10.

The substrate is transferred to a 30 mL reagent container and placed in that reagent rack at position 2 (holder 1).

The alpha-glucosidase help reagent is transferred to a 50 mL reagent container and placed in the reagent rack at position 2 (holder C)

8.3 Cobas Fare Analysis

The main principles of the analysis are as follows:

20 μL sample and 10μ rinse-water are pipetted into the cuvette along with 250 μL alpha-glucosidase help reagent. The cuvette rotates for 10 seconds and the reagents are thrown out into the horizontal cuvettes. 25 μL substrate and 20 μL rinse-water are pipetted off. After a 1 second wait to ensure that the temperature is 37° C., the cuvette rotates again and the substrate is mixed into the horizontal cuvettes. Absorbance is measured for the first time after 120 seconds and then every 5 seconds. Absorbance is measured a total of 37 times for each sample.

9. Calculations

The activity of the samples is calculated relative to Novo Nordisk A/S standard.

The standard curve is plotted by the analyzer. The curve is to be gently curved, rising steadily to an absorbance of around 0.25 for standard no. 5.

The activity of the samples in KNU(S)/mL is read off the standard curve by the analyzer.

The final calculations to allow for the weights/dilutions used employ the following formula:

Activity in KNU(S)/g=S×V×F/W

S=analysis result read off (KNU(S)/mL V=volume of volumetric flask used in mL F=dilution factor for second dilution W=weight of enzyme sample in g

9.2 Calculation of Mean Values

Results are stated with 3 significant digits. However, for sample activity <10 KNU(S)/g, only 2 significant digits are given.

The following rules apply on calculation of mean values:

1. Data which deviates more than 2 standard deviations from the mean value is not included in the calculation. 2. Single and double determination over one run:

The mean value is calculated on basis of results lying within the standard curve's activity area.

3. Double determinations over two runs: All values are included in the mean value. Outliers are omitted.

10. Accuracy and Precision

The coefficient of variation is 2.9% based on retrospective validation of analysis results for a number of finished products and the level control.

Phadebas Assay (for Alpha-Amylase Activity Determination)

Alpha-amylase activity is determined by a method employing Phadebas® tablets as substrate. Phadebas tablets (Phadebas® Amylase Test, supplied by Pharmacia Diagnostic) contain a cross-linked insoluble blue-colored starch polymer which has been mixed with bovine serum albumin and a buffer substance and tabletted.

For every single measurement one tablet is suspended in a tube containing 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, 0.1 mM CaCl₂, pH adjusted to the value of interest with NaOH). The test is performed in a water bath at the temperature of interest. The alpha-amylase to be tested is diluted in x ml of 50 mM Britton-Robinson buffer. 1 ml of this alpha-amylase solution is added to the 5 ml 50 mM Britton-Robinson buffer. The starch is hydrolyzed by the alpha-amylase giving soluble blue fragments. The absorbance of the resulting blue solution, measured spectrophotometrically at 620 nm, is a function of the alpha-amylase activity.

It is important that the measured 620 nm absorbance after 10 or 15 minutes of incubation (testing time) is in the range of 0.2 to 2.0 absorbance units at 620 nm. In this absorbance range there is linearity between activity and absorbance (Lambert-Beer law). The dilution of the enzyme must therefore be adjusted to fit this criterion. Under a specified set of conditions (temp., pH, reaction time, buffer conditions) 1 mg of a given alpha-amylase will hydrolyze a certain amount of substrate and a blue colour will be produced. The colour intensity is measured at 620 nm. The measured absorbance is directly proportional to the specific activity (activity/mg of pure alpha-amylase protein) of the alpha-amylase in question under the given set of conditions.

Alternative Alpha-Amylase Activity Method (PNP-G7 Assay)

Alpha-amylase activity is determined by a method employing the PNP-G7 substrate. PNP-G7 which is a abbreviation for p-nitrophenyl-alpha,D-maltoheptaoside is a blocked oligosaccharide which can be cleaved by an endo-amylase. Following the cleavage, the alpha-glucosidase included in the kit digest the substrate to liberate a free PNP molecule which has a yellow colour and thus can be measured by visible spectophometry at λ=405 nm (400-420 nm). Kits containing PNP-G7 substrate and alpha-glucosidase is manufactured by Boehringer-Mannheim (cat. no. 1054635).

To prepare the substrate one bottle of substrate (BM 1442309) is added to 5 ml buffer (BM1442309). To prepare the alpha-glucosidase one bottle of alpha-glucosidase (BM 1462309) is added to 45 ml buffer (BM1442309). The working solution is made by mixing 5 ml alpha-glucosidase solution with 0.5 ml substrate.

The assay is performed by transforming 20 μl enzyme solution to a 96 well microtiter plate and incubating at 25° C. 200 μl working solution, 25° C. is added. The solution is mixed and pre-incubated 1 minute and absorption is measured every 15 sec. over 3 minutes at OD 405 nm.

The slope of the time dependent absorption-curve is directly proportional to the specific activity (activity per mg enzyme) of the alpha-amylase in question under the given set of conditions.

EXAMPLES Example 1 Construction of Variants of BSG Alpha-Amylase (SEQ ID NO: 3)

The gene encoding BSG, amyS, is located in plasmid pPL1117. This plasmid contains also the gene conferring resistance towards kanamycin and an origin of replication, both obtained from plasmid pUB110 (Gryczan et al., 1978, J. Bact. 134:318-329).

The DNA sequence of the mature part of amyS is shown as SEQ ID NO: 11 and the amino acid sequence of the mature protein is shown as SEQ ID NO: 3

BSG variant TVB145, which contains a deletion of 6 nucleotides corresponding to amino acids I181-G182 in the mature protein, is constructed as follows:

Polymerase Chain Reaction (PCR) is utilized to amplify the part of the amyS gene (from plasmid pPL1117), located between DNA primers BSG1 (SEQ ID NO: 16) and BSGM2 (SEQ ID NO: 19). BSG1 is identical to a part of the amyS gene whereas BSGM2 contains the 6 bp nucleotide deletion. A standard PCR reaction is carried out: 94° C. for 5 minutes, 25 cycles of (94° C. for 45 seconds, 50° C. for 45 seconds, 72° C. for 90 seconds), 72° C. for 7 minutes using the Pwo polymerase under conditions as recommended by the manufacturer, Boehringer Mannheim Gmbh.

The resulting approximately 550 bp amplified band was used as a megaprimer (Barik and Galinski, 1991, Biotechniques 10: 489-490) together with primer BSG3 in a second PCR with pPL1117 as template resulting in a DNA fragment of approximately 1080 bp.

This DNA fragment is digested with restriction endonucleases Acc65I and SalI and the resulting approximately 550 bp fragment is ligated into plasmid pPL1117 digested with the same enzymes and transformed into the protease- and amylase-deleted Bacillus subtilis strain SHA273 (described in WO 92/11357 and WO 95/10603).

Kanamycin resistant and starch degrading transformants were analysed for the presence of the desired mutations (restriction digest to verify the introduction of a HindIII site in the gene). The DNA sequence between restriction sites Acc65I and SalI was verified by DNA sequencing to ensure the presence of only the desired mutations.

BSG variant TVB146 which contains the same 6 nucleotide deletion as TVB145 and an additional substitution of asparagine 193 for a phenylalanine, N193F, was constructed in a similar way as TVB145 utilizing primer BSGM3 (SEQ ID NO: 20) in the first PCR.

BSG variant TVB161, containing the deletion of I181-G182, N193F, and L204F, is constructed in a similar way as the two previous variants except that the template for the PCR reactions is plasmid pTVB146 (pPL1117 containing the TVB146-mutations within amyS and the mutagenic oligonucleotide for the first PCR is BSGM3.

BSG variant TVB162, containing the deletion of I181-G182, N193F, and E210H, is constructed in a similar way as TVB161 except that the mutagenic oligonucleotide is BSGM4 (SEQ ID NO: 21).

BSG variant TVB163, containing the deletion of I181-G182, N193F, and E214Q, is constructed in a similar way as TVB161 except that the mutagenic oligonucleotide is BSGM5 (SEQ ID NO: 22).

The above constructed BSG variants were then fermented and purified as described above in the “Material and Methods” section.

Example 2 Measurement of the Calcium- and pH-Dependent Stability

Normally, the industrial liquefaction process runs using pH 6.0-6.2 as liquefaction pH and an addition of 40 ppm free calcium in order to improve the stability at 95° C.-105° C. Some of the herein proposed substitutions have been made in order to improve the stability at

1. lower pH than pH 6.2 and/or 2. at free calcium levels lower than 40 ppm free calcium.

Two different methods have been used to measure the improvements in stability obtained by the different substitutions in the alpha-amylase from B. stearothermophilus:

Method 1. One assay which measures the stability at reduced pH, pH 5.0, in the presence of 5 ppm free calcium.

10 μg of the variant were incubated under the following conditions: A 0.1 M acetate solution, pH adjusted to pH 5.0, containing 5 ppm calcium and 5% w/w common corn starch (free of calcium). Incubation was made in a water bath at 95° C. for 30 minutes.

Method 2. One assay which measure the stability in the absence of free calcium and where the pH is maintained at pH 6.0. This assay measures the decrease in calcium sensitivity:

10 μg of the variant were incubated under the following conditions: A 0.1 M acetate solution, pH adjusted to pH 6.0, containing 5% w/w common corn starch (free of calcium). Incubation was made in a water bath at 95° C. for 30 minutes.

Stability Determination

All the stability trials 1, 2 have been made using the same set up. The method was: The enzyme was incubated under the relevant conditions (1-4). Samples were taken at 0, 5, 10, 15 and 30 minutes and diluted 25 times (same dilution for all taken samples) in assay buffer (0.1 M 50 mM Britton buffer pH 7.3) and the activity was measured using the Phadebas assay (Pharmacia) under standard conditions pH 7.3, 37° C.

The activity measured before incubation (0 minutes) was used as reference (100%). The decline in percent was calculated as a function of the incubation time. The table shows the residual activity after 30 minutes of incubation.

Stability Method 1. /Low pH Stability Improvement

SEQ. ID NO: 3 SEQ. ID NO: 3 SEQ. ID NO: 3 VARIANT WITH VARIANT WITH WT. VARIANT WITH DELETION IN DELETION IN SEQ. ID. NO: 3 DELETION IN POS. I181-G182 + POS. I181-G182 + MINUTES OF AMYLASE POS. I181-G182 N193F N193F + E214Q INCUBATION (BSG) (TVB145) (TVB146) (TVB163) 0 100 100 100 100 5 29 71 83 77 10 9 62 77 70 15 3 50 72 67 30 1 33 62 60

Stability Method 1. /Low pH Stability Improvement

The temperature described in method 1 has been reduced from 95° C. to 70° C. since the amylases mentioned for SEQ ID NO: 1 and 2 have a lower thermostability than the one for SEQ ID NO: 3.

SEQ. ID NO: 2 SEQ. ID NO: 1 WT. VARIANT WITH VARIANT WITH MINUTES OF SEQ. ID. NO: 2 DELETION IN SEQ. ID NO: 1 DELETION IN INCUBATION AMYLASE POS. D183-G184 AMYLASE POS. T183-G184 0 100 100 100 100 5 73 92 41 76 10 59 88 19 69 15 48 91 11 62 30 28 92 3 59

Stability Method 2. /Low Calcium Sensitivity

SEQ ID NO: 3 SEQ ID NO: 3 SEQ ID NO: 3 VARIANT WITH VARIANT WITH WT. VARIANT WITH DELETION IN DELETION IN SEQ ID NO: 3 DELETION IN POS. I181-G182 + POS. I181-G182 + MINUTES OF AMYLASE POS. I181-G182 N193F N193F + E214Q INCUBATION (BSG) (TVB145) (TVB146) (TVB163) 0 100 100 100 100 5 60 82 81 82 10 42 76 80 83 15 31 77 81 79 30 15 67 78 79

Specific Activity Determination.

The specific activity was determined using the Phadebas assay (Pharmacia) as activity/mg enzyme. The activity was determined using the alpha-amylase assay described in the Materials and Methods section herein.

The specific activity of the parent enzyme and a single and a double mutation was determined to:

BSG: SEQ ID NO: 3 (Parent enzyme) 20000 NU/mg TVB145: SEQ ID NO: 3 with the deletion in positions I181-G182: (Single mutation) 34600 NU/mg TVB146: SEQ ID NO: 3 with the deletion in positions I181-G182 + N193F: (Double mutation) 36600 NU/mg TVB163: SEQ ID NO: 3 with the deletion in positions I181-G182+N193F+E214Q: (Triple mutation) 36300 NU/mg

Example 3 Pilot Plant Jet Cook and Liquefaction with Alpha-Amylase Variant TVB146

Pilot plant liquefaction experiments were run in the mini-jet system using a dosage of 50 NU (S)/g DS at pH 5.5 with 5 ppm added Ca⁺⁺, to compare the performance of formulated BSG alpha-amylase variant TVB146 (SEQ ID NO: 3 with deletion in positions I181-G182+N193F) with that of parent BSG alpha-amylase (SEQ ID NO: 3). The reaction was monitored by measuring the DE increase (Neocuproine method) as a function of time.

Corn starch slurries were prepared by suspending 11.8 kg Cerestar C*Pharm GL 03406 (89% starch) in deionized water and making up to 30 kg. The pH was adjusted to 5.5 at ambient temperature, after the addition of 0.55 g CaCl₂. 2H₂O.

The following enzymes were used:

TVB146 108 KNU(S)/g, 146 KNU(SM9)/g BSG amylase 101 KNU(S)/g, 98 KNU(SM9)/g

An amount of enzyme corresponding to 50 NU (SM9)/g DS was added, and the conductivity adjusted to 300 mS using NaCl. The standard conditions were as follows:

Substrate concentration 35% w/w (initial) 31.6-31.9% w/w (final) Temperature 105° C., 5 minutes (Primary liquefaction) 95° C., 90 minutes (Secondary liquefaction) pH (initial) 5.5

After jetting, the liquefied starch was collected and transported in sealed thermos-flasks from the pilot plant to the laboratory, where secondary liquefaction was continued at 95° C.

10 ml samples were taken at 15 minute intervals from 15-90 minutes. 2 drops of 1 N HCl were added to inactivate the enzyme. From these samples, 0.3-0.1 g (according to the expected DE) were weighed out and diluted to 100 ml. Reducing sugars were then determined according to the Neocuproine method (Determination of reducing sugar with improved precision. Dygert et al., 1965, Anal. Biochem. 13: 368) and DE values determined. The development of DE as a function of time is given in the following table:

Time TVB146 BSG (min.) DE (neocuproine) 15 2.80 2.32 30 4.88 3.56 45 6.58 4.98 60 8.17 6.00 75 9.91 7.40 90 11.23 8.03

As can be seen the alpha-amylase variant TVB146 performed significantly better under industrially relevant application conditions at low levels of calcium than the parent BSG alpha-amylase.

Example 4 Jet Cook and Liquefaction with a Combination of Alpha-Amylase Variants (TVB146 and LE174)

Jet cook and liquefaction using a combination of the alpha-amylase variants, TVB146 and LE174 (ratio 1:1) were carried out at the following conditions:

Substrate A.E. Staley food grade powdered corn starch (100 lbs) D.S. 35% using DI water Free Ca²⁺ 2.7 ppm at pH 5.3 (none added, from the starch only) Initial pH 5.3 Dose AF9 units (AF9 is available on request) for each enzyme variant was 28 NU/g starch db for a total dose of 56 NU/g Temperature in primary liquefaction 105° C. Hold time in primary liquefaction 5 minutes Temperature in secondary liquefaction 95° C.

At 15 minutes into secondary liquefaction 1.5 gms of hydrolyzate was added to a tared one liter volumetric containing 500 cc of DI water and 1 ml of one normal HCl and the exact wt. added was recorded. This was repeated at 15 minute intervals out to 90 minutes with an additional point at 127 minutes. These were diluted to one liter and determined for dextrose equivalence via Neocuproine method as described by Dygert et al., 1965, Determination of reducing sugar with improved precision, Anal. Biochem. 13: 368.

The results were as follows:

Time DE 15 3.2 30 4.8 45 6.3 60 7.8 75 9.4 90 10.4 127 13.1

Example 5 Isolation of Genomic DNA from DSM 12648 and DSM 12649

The strains Bacillus sp. DSM 12649 (the AA560 alpha-amylase) and Bacillus sp. DSM 12648 (the AA349 alpha-amylase) were propagated in liquid TY medium (as described in Ausubel et al. (1995)). After 16 hours incubation at 37° C. and 300 rpm, the cells were harvested, and genomic DNA isolated by the method described by Pitcher et al. (1989).

Genomic Library Construction

Genomic DNA of strain DSM 12649 was partially digested with restriction enzyme Sau3A, and size-fractionated by electrophoresis on a 0.7% agarose gel. Fragments between 2 and 10 kb in size were isolated by electrophoresis onto DEAE-cellulose paper (Dretzen et al. (1981).

Isolated DNA fragments were ligated to BamHI digested pSJ1678 plasmid DNA, and the ligation mixture was used to transform E. coli SJ2.

Transformation

E. coli SJ2 host cells were prepared for and transformed by electroporation using a gene PULSER™ electroporator from BIO-RAD as described by the supplier.

Identification of Positive Transformant:

A DNA library in E. coli SJ2, constructed as described above, was screened on LB agar plates (described in Ausbel et al. (1995)) containing 0.5% AZCL-amylose (Megazyme) and 10 ig/ml Chloramphenicol and incubated overnight at 37° C. Clones expressing amylase activity appeared with blue diffusion haloes. One such clone was named LiH1274. The DNA was further characterized by DNA sequencing of part of the cloned Sau3A DNA fragment.

Example 6 Determination of the DNA Sequence of the Gene Encoding Alpha-Amylase from Strain DSM 12648 (AA349)

The clone constituting a large chromosomal fragment containing the gene encoding the amylolytic activity inserted into plasmid pSJ1678, pLiH1247, was used as template to specifically PCR amplify internal DNA fragments of the alpha-amylase encoding gene by the use of degenerate primers directed towards the conserved regions in known Bacillus alpha-amylases.

The degenerate primers were directed towards the following regions/amino acid sequences:

For36:  (SEQ ID NO: 27) GITA(L/V/I)W(I/L)  For97:  (SEQ ID NO: 28) VY(G/A)D(V/F/L)V(M/L/I/F)NH  For227:  (SEQ ID NO: 29) DG(F/I)R(F/L/I/V)DA(A/V)KH  Rev235:  (SEQ ID NO: 30) DG(F/I)R(F/L/I/V)DA(A/V)KH  Rev328:  (SEQ ID NO: 31) VTFV(D/E)NHD  Rev410:  (SEQ ID NO: 32) GWTREG 

The various combinations of forward (For) and reverse (Rev) primers were used in PCR and internal DNA fragments could be amplified.

The DNA fragments were purified by QIAquick spin colums (QUIGEN) and sequenced utilizing the same degenerate primers.

From sequence the DNA sequence (SEQ ID NO: 23) of the complete coding region encoding the mature AA349 alpha-amylase (SEQ ID NO: 26) was determined by a standard primers-walking approach.

Example 7 Determination of the DNA Sequence of the Gene Encoding Alpha Amylase from Strain DSM 12649 (AA560)

A preparation of chromosomal DNA from strain DSM 12649 was utilized as template in a similar experiment to the one described above in Example 7 in order to determine the DNA sequence of the AA560 alpha-amylase (SEQ ID NO: 24).

Example 8 Subcloning of the AA349 Alpha-Amylase into pTVB110

pTVB110 is a plasmid replicating in Bacillus subtilis by the use of origin of replication from pUB110 (Gryczan, 1978, J. Bact. 134:318-329). The plasmid further encodes the cat gene, conferring resistance towards chlorampenicol, obtained from plasmid pC194 (Horinouchi and Weisblum, 1982, J. Bact. 150: 815-825). The plasmid harbors a truncated version of the Bacillus licheniformis alpha-amylase gene, amyL, such that the amyL promoter, signal sequence and transcription terminator are present, but the plasmid does not provide an amy-plus phenotype (halo formation on starch containing agar).

In order to express high amount of the AA349 alpha-amylase the mature gene was fused precisely to the amyL signal sequence so that transcription is initiated by the amyL promoter and translocation is directed by the amyL signal sequence.

A PstI site is found within the mature AA349 alpha-amylase. Since the cloning of the gene into pTVB110 would utilize the PstI site in pTVB110, the PstI site located within the AA349 alpha-amylase gene was destroyed during the cloning (by introduction of a silent mutation for amino acid Alanine 88 (GCA to GCG).

Primers 188cloningN and 188(Pst-) were used to amplify an approximately 280 bp fragment by PCR on plasmid pLiH1247 using the Pwo polymerase under conditions recommended by the manufacturer (Boehringer Mannheim). This fragment was purified from agarose gel and used as a megaprimer (Sarkar and Sommer, 1990, Biotechniques 8: 404-407) together with primer 188cloningC to amplify the full length gene encoding the mature amylase in a second PCR.

The resulting approximately 1480 bp fragment was digested with restriction endonucleases PstI and SfiI and ligated with plasmid pTVB110 digested with the same enzymes.

Protease and amylase deleted Bacillus subtilis strain SHa273 (mentioned in WO 95/10603) was transformed with the ligation mixture and the DNA sequence of an amy-plus transformant was verified. This plasmid is denoted pTVB231.

Oligonucleotides:

188(Pst-): (SEQ ID NO: 33) 5′ GGC GTT AAC CGC AGC TTG TAA C 188cloningC: (SEQ ID NO: 34) 5′ CCG AGC TCG GCC GGC TGG GCC GTC GAC TTA TTT GTT TAC CCA AAT AGA AAC  188cloningN: (SEQ ID NO: 35) 5′ CAT TCT GCA GCA GCG GCG CAC CAT AAT GGT ACG AAC G

Example 9 Subcloning of the AA560 Alpha-Amylase into pTVB110

DNA sequencing revealed a high DNA identity between alpha-amylases from stains DSM12648 (AA349) and DSM 12649 (AA560). Consequently the same oligonucleotides and strategy was utilized for the cloning of AA560 alpha-amylase into expression vector pTVB110 resulting in plasmid pTVB232, which was then fermented using standard techniques.

Example 10 Purification of the AA560 Alpha-Amylase

The culture broth was flocculated by adding 0.01 ml 50% (w/w) CaCl₂, 2H₂O, 0.0125 ml 12% (w/w) Sodium aluminate, 0.025 ml 10% C521 and 0.075 ml 0.1% A130 pr. ml culture broth. A clear solution was obtained after centrifugation. The enzyme solution was added ammonium sulphate to a final concentration of 1.2 M and applied on a Butyl Toyo Pearl column (100 ml) previously equilibrated in 1.2 M ammonium sulphate, 10 mM Tris-HCl, pH 7.0. The amylase was eluted using 5 mM Tris-HCl, pH 7.0 and the eluted pool was dialysed against 5 mM Tris-HCl over night. The fraction was then subjected to ion exchange chromatography using a Q-Sepharose column (200 ml) previously equilibrated in 20 mM Tris-HCl, pH 9.0. Unbound material was washed out with the equilibration buffer, and the amylase was eluted using a linear gradient 0-1 M NaCl, 20 mM Tris-HCl, pH 9.0. Purity of the amylase preparation was above 95% judged by SDS-PAGE.

Example 11 Characterization of the AA560 Alpha-Amylase

The alpha-amylase activity was measured using both the Phadebas assay (37° C., pH 7.3) and the Alternative pNPG7 Assay (25° C., pH 7.1) described above. pH- and temperature profiles were made at selected pH- and temperature values. The pH-profile was measured at 37° C. and the temperature profile was measured at pH 9.0

Isoelectric Point was determined using isoelectric focusing (Pharmacia, Ampholine, pH 3.5-9.3).

TABLE 1 Specific activity and pI. Specific activity NU/ml NU/ml Enzyme Phadebas pNPG7 pI AA560 (SEQ ID NO: 4) 35000 6000 7-8 SP722 (SEQ ID NO: 2) 35000 6000 7-9 SP690 (SEQ ID NO: 1) 35000 7000 5-6 E = 3.2 cm⁻¹ *(g/l)⁻¹ for AA560, SP722 and SP690

The result of the pH-optimum determination and temperature optimum determination is shown in FIG. 2 and FIG. 3, respectively.

Example 12 Washing Test

Washing performance was evaluated by washing soiled test swatches for 15 and 30 minutes at 25° C. and 40° C., respectively, in detergent solutions with the AA560 alpha-amylase of the invention.

The detergents used are disclosed in Table 2 below. The A/P Model Detergent is described in the Materials section above. The other detergents are commercially available detergents. Commercial detergents containing amylase were inactivated by microwaves before wash.

The purified recombinant AA560 alpha-amylase of Example 6 was added to the detergent solutions at the concentration indicated below. The test swatches were soiled with orange rice starch (CS-28 swatches available from CFT, Center for Test Material, Holland). After washing, the swatches were evaluated by measuring the remission at 460 nm using a Elrepho Remission Spectrophotometer. The results are expressed as ΔR=remission of the swatch washed with the alpha-amylase minus the remission of a swatch washed at the same conditions without the alpha-amylase.

TABLE 2 Detergents and wash conditions. Det. Enzyme Water Dose Inacti- dose Temp. Time hardness Area Detergent g/l vation mg/l ° C. min pH °dH Ca:Mg A/P Model 3 − 1 25 15 10.5 6 2:1 detergent 97 Latin Omo Multi 3 − 1 25 15 10.6 6 2:1 America Acao Europe Omo conc. 4 + 0.2 40 30 10.2 15 4:1 Powder Europe Ariel Futur 5 + 0.2 40 30 9.0 15 4:1 liquid

The results are shown in FIGS. 4-7. The results demonstrate that the alpha-amylase of the invention is effective in both detergents at highly alkaline pH.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

REFERENCES CITED

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1. A variant of a parent Termamyl-like α-amylase with α-amylase activity comprising mutations in two, three, four, five or six of the following regions/positions or in corresponding positions in other parent Termamyl-like α-amylases: (relative to SEQ ID NO: 1): 1: R181*,G182*,T183*,G184* 2: N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 3: V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y; 4: E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 5: E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 6: K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V; (relative to SEQ ID NO: 2): 1: R181*,G182*,D183*,G184* 2: N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 3: V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y; 4: E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 5: E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 6: K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V; (Relative to SEQ ID NO: 3): 1: R179*,G180,I181*,G182* 2: N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 3: L204A,R,D,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V; 4: E210A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 5: E214A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 6: S267A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V Relative to SEQ ID NO: 4): 1: N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 2: I201A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V; 3: D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 4: E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 5: Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V; (relative to SEQ ID NO: 5): 1: R176*,G177*,E178,G179* 2: N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 3: V201A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y; 4: D207A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 5: E211A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 6: Q264A,R,D,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V; (relative to SEQ ID NO: 6): 1: R181*,G182*,H183*,G184* 2: N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 3: I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V; 4: E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 5: E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; 6: K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
 2. The variant according to claim 1, comprising the following mutations: N190F/Q264S in SEQ ID NO: 4 or in corresponding positions in another parent α-amylase.
 3. The variant according to claim 1, comprising the following mutations: I181*/G182*/N193F in SEQ ID NO: 3 or in corresponding positions in another parent Termamyl like α-amylase.
 4. The variant according to claim 3, further comprising a substitution in position E214Q in SEQ ID NO: 3 or in a corresponding position in another parent Termamyl like α-amylase.
 5. The variant according to claim 1, wherein the parent α-amylase is a hybrid α-amylase of SEQ ID NO: 4 and SEQ ID NO:
 5. 6. The variant according to claim 5, wherein the parent hybrid α-amylase is a hybrid alpha-amylase comprising the 445 C-terminal amino acid residues of the B. licheniformis α-amylase shown in SEQ ID NO: 4 and the 37 N-terminal amino acid residues of the α-amylase derived from B. amyloliquefaciens shown in SEQ ID NO:
 5. 7. The variant according to claim 6, wherein the parent hybrid Termamyl-like α-amylase further has the following mutations: H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 4).
 8. The variant according to claim 1, exhibiting increased stability at acidic pH and/or low Ca²⁺ concentration:
 9. A DNA construct comprising a DNA sequence encoding an α-amylase variant according to claim
 1. 10. A recombinant expression vector which carries a DNA construct according to claim
 9. 11. A cell which is transformed with a DNA construct according to claim 9 or a vector according to claim
 10. 12. A cell according to claim 11, which is a microorganism.
 13. A cell according to claim 12, which is a bacterium or a fungus.
 14. The cell according to claim 13, which is a grampositive bacterium such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus or Bacillus thuringiensis.
 15. A detergent additive comprising an α-amylase variant according to claim 1, optionally in the form of a non-dusting granulate, stabilised liquid or protected enzyme.
 16. A detergent additive according to claim 15 which contains 0.02-200 mg of enzyme protein/g of the additive.
 17. A detergent additive according to claim 15, which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, another amylolytic enzyme and/or a cellulase.
 18. A detergent composition comprising an α-amylase variant according to claim
 1. 19. The detergent composition according to claim 18 which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, another amylolytic enzyme and/or a cellulase.
 20. A manual or automatic dishwashing detergent composition comprising an α-amylase variant according to claim
 1. 21. A dishwashing detergent composition according to claim 20 which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, another amylolytic enzyme and/or a cellulase.
 22. A manual or automatic laundry washing composition comprising an α-amylase variant according to claim
 1. 23. A laundry washing composition according to claim 22, which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, an amylolytic enzyme and/or a cellulase.
 24. A composition comprising: (i) a mixture of the α-amylase from B. licheniformis having the sequence shown in SEQ ID NO: 4 with one or more variants according to claim 1 derived from (as the parent Termamyl-like α-amylase) the B. stearothermophilus α-amylase having the sequence shown in SEQ ID NO: 3; or (ii) a mixture of the α-amylase from B. stearothermophilus having the sequence shown in SEQ ID NO: 3 with one or more variants according to claim 1 derived from one or more other parent Termamyl-like α-amylases; or (iii) a mixture of one or more variants according claim 1 derived from (as the parent Termamyl-like α-amylase) the B. stearothermophilus α-amylase having the sequence shown in SEQ ID NO: 3 with one or more variants according to the invention derived from one or more other parent Termamyl-like α-amylases.
 25. A composition comprising: a mixture of one or more variants according claim 1 derived from (as the parent Termamyl-like α-amylase) the B. stearothermophilus α-amylase having the sequence shown in SEQ ID NO: 3 and a Termamyl-like alpha-amylase derived from the B. licheniformis α-amylase having the sequence shown in SEQ ID NO:
 4. 26. The composition comprising: a mixture of one or more variants according claim 1 derived from (as the parent Termamyl-like α-amylase) the B. stearothermophilus α-amylase having the sequence shown in SEQ ID NO: 3 and a hybrid alpha-amylase comprising a part of the B. amyloliquefaciens α-amylase shown in SEQ ID NO: 5 and a part of the B. licheniformis α-amylase shown in SEQ ID NO:
 4. 27. The composition according to claim 26, wherein the hybrid α-amylase is a hybrid alpha-amylase comprising the 445 C-terminal amino acid residues of the B. licheniformis α-amylase shown in SEQ ID NO: 4 and the 37 N-terminal amino acid residues of the α-amylase derived from B. amyloliquefaciens shown in SEQ ID NO:
 5. 28. The composition according to claim 27, wherein the hybrid α-amylase further has the following mutations: H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 4).
 29. The composition according to claim 26, comprising a mixture of TVB146 and LE174.
 30. A method for generating a variant of a parent Termamyl-like α-amylase, which variant exhibits increased stability at low pH and at low calcium concentration relative to the parent, the method comprising: (a) subjecting a DNA sequence encoding the parent Termamyl-like α-amylase to random mutagenesis, (b) expressing the mutated DNA sequence obtained in step (a) in a host cell, and (c) screening for host cells expressing a mutated α-amylase which has increased stability at low pH and low calcium concentration relative to the parent α-amylase. 